Although there are not many works tackling the problem of the discrimination between children and adult faces, current solutions are focused in controlled environments which decreases the number of applications to use this kind of models. The proposed model archived a higher accuracy with the reference models [2], [4] fitting a set of 68 facial landmarks to the output of the recognition model to improving the measurement of the face and the classification rates. On the other hand, a limitation of the model is that the body and the face must be present in the image, a possible way to tackle this limitation may be to use an algorithm to recognize heads instead of a face recognition algorithm.

61% 78% 64% **86.4%** 74.6%

In conclusion, the results suggest that it's possible to improve discrimination between children and adult faces measuring the head by using facial landmarks instead of the bounding boxes provided by a facial

VI. ACKNOWLEDGMENTS

This research has been funded by project "Analysis and classification techniques of voice and facial expressions: application to neurological diseases in newborns and adults" from AMEXCID, supported by

REFERENCES [1] K. Lee, C. Lee, S. A. Kim, and Y. H. Kim, "Fast object detection based on color histograms and local binary patterns," IEEE Reg. 10 Annu. Int. Conf.

[2] M. B. Hans Weda, "AUTOMATIC CHILDREN DETECTION IN DIGITAL IMAGES," pp. 1687–1690,

[3] W.-B. Horng, C.-P. Lee, and C.-W. Chen, "Classification of Age Groups Based on Facial Features," Tamkang J. Sci. Eng., vol. 4, no. 3, pp. 183–

[4] O. F. Ince, I. F. Ince, J. S. Park, J. K. Song, and B. W. Yoon, "Child and adult classification using biometric features based on video analytics," ICIC Express Lett. Part B Appl., vol. 8, no. 5, pp. 819–825,

[5] B. Yang, J. Yan, Z. Lei, and S. Z. Li, "Aggregate channel features for multi-view face detection," IJCB 2014 - 2014 IEEE/IAPR Int. Jt. Conf. Biometrics, 2014. [6] T. Surasak, I. Takahiro, C. H. Cheng, C. E. Wang, and P. Y. Sheng, "Histogram of oriented gradients for human detection in video," Proc. 2018 5th Int. Conf. Bus. Ind. Res. Smart Technol. Next Gener. Information, Eng. Bus. Soc. Sci. ICBIR 2018, pp. 172–176, 2018. [7] P. Viola and M. Jones, "Rapid Object Detection using a Boosted Cascade of Simple Features," Surf. Sci.,

[8] F. De La Torre, W. S. Chu, X. Xiong, F. Vicente, X. Ding, and J. Cohn, "IntraFace," 2015 11th IEEE Int. Conf. Work. Autom. Face Gesture Recognition, FG

[9] O.-E. F. Morales-Vargas E., Reyes-García C.A., Peregrina-Barreto H., "Facial expression recognition with fuzzy explainable models," Model. Anal. Vocal

[10] O. F. Ince, J. S. Park, J. Song, and B. W. Yoon, "Child and Adult Classification Using Ratio of Head and Body Heights in Images," Int. J. Comput. Commun.

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BHR [10] BHR+GF BLHR **BLHR+GF** BNLHR

methodology.

This work analyzes the application of sonification techniques to EEG signals for an almost real time assessment of epileptic seizures onset. Sonification concerns the transformation of numerical data into acoustic signals. EEG sonification could speed up its interpretation and reduce the amount of visual information to be analyzed. The dataset consists of 24 EEG recordings coming from 22 children with absence seizures, clinically evaluated at the Meyer Children's Hospital in Firenze, Italy. The obtained low false positive rate (<1%), shows the usefulness of EEG sonification techniques in supporting automatic and early seizure detection.

Keywords: sonification, EEG, epilepsy, patientspecific, entropy.

### I. INTRODUCTION

Epilepsy is one of the main brain disorders, affecting about 1% of the world population [1]. It occurs mainly in childhood and increases, in terms of cumulative prevalence, in the elderly population [2]. Epilepsy, an enduring predisposition to manifest epileptic seizures, is also defined as "a transient occurrence of signs and/or symptoms due to abnormal excessive and synchronous neuronal activity in the brain." [3], [4].

Given their high variability both in terms of aetiology, clinical signs, and electrophysiological characteristics, epileptic seizures are divided into several categories [5].

Electroencephalography (EEG) is one of the main technology supporting the diagnosis of epilepsy.

The work presented here focuses on absence seizures, a type of generalized non-motor epileptic seizures [6]. The EEG signal of a patient with a typical absence shows generalized spike-wave discharges at 3Hz.

One of the main technological problems in the analysis of EEG signals in epilepsy is the presence of artefacts that, summed up to the data to be analyzed, can make the analysis in real time complex and burdensome.

In recent years there has been a growing interest in techniques allowing a near real-time clinical assessment of seizures (i.e. low latency times) [7, 8, 9], especially sonification. According to [10], sonification is defined as: "the transformation of data relations into perceived relations in an acoustic signal for the purposes of facilitating communication or interpretation".

Sonification can be used for performing a fast detection of clinical events or monitoring clinical information. Recently, Schwarz et al. [11] presented a psychoacoustic sonification technique for real time monitoring of neonatal oxygen saturation levels.

G.I. Mihalas et al. [12] gave several examples of physiological signal sonification, such as ECG, Heart Rate etc., and discussed their biomedical applications.

F.B. Vialatte et al. [13] used sonification techniques to discriminate EEGs of patients with mild cognitive impairment from those of healthy control subject.

Our recent paper [9] focused on techniques applied to the EEG signal for early seizure detection, along with artificial intelligence methods for the automatic recognition of absence seizures.

In the present work, the sonification techniques presented in [9] are analysed, with the purpose of reducing the false positive rates in the sonified EEG signals.

Patient-specific techniques are proposed to remove background noise from the sonified EEG signal. Moreover, methods are suggested for enhancing amplitude and patterns related to epileptic seizures or possible artefacts. Finally, similarly to [11], a statistical evaluation of the sonified EEG signal is presented, taking into account relevant differences between false

Claudia Manfredi (edited by), *Models and Analysis of Vocal Emissions for Biomedical Applications : 11th International Workshop, December, 17-19, 2019*, © 2019 FUP, CC BY 4.0 International, published by Firenze University Press (www.fupress.com), ISSN 2704-5846 (online), ISBN (online PDF) 978-88-6453-961-4

positives not discarded by the automatic recognition process and the true epileptic seizures.

### II. METHODS

To compare the performance of the proposed methods, the same EEG recordings used as test set in [9] and the same automatic recognition algorithms are considered here. The test set consists of 24 recordings of 22 children (7 males, 15 females, and age range 4 – 18 years) with absence seizures evaluated at the Paediatric Neurology Unit, Children's Hospital A. Meyer, Firenze, Italy. The duration of the signals is 47 ± 10 min with sampling frequency fs=256Hz. Written informed consent was obtained from all patients or their guardians.

The proposed methods are implemented under MATLAB software (version 2017b [14]) installed on a Hp Pavillion 15 notebook (OS Windows 10, 64 bit) Intel Core i7-5500U processor, CPU 2.40 GHz, RAM 16 Gb.

In [9] the automatic recognition gave 1% average percentage of false positives (FPR metric, ONLINE method) in the sonified signal. Here possible techniques that could further reduce false positives in the sonified signal are investigated.

The sonification procedure proposed in [9] is based on the following relationship:

### [ABS – ABS – ABS] → [beep1beep2 → soundabs] (1)

Eq. (1) is described as follows. If the automatic recognition step detects three (or more) consecutive absences (ABS), the first 2 consecutive seconds will produce 2 beeps (beep1 and beep2) as pre-alarms. This sound represents a first discrimination between a possible absence and a false positive.

As described in detail in [9], the following seconds classified as ABS produce the soundabs. This is the sonified signal of the cumulated sum of the 6th levels of decomposition of the EEG signals obtained applying the Stationary Wavelet Transform.

False positives are grouped into: background without intercritical activity, intercritical activity or, more generally, elements of electrophysiological interest of duration <2s, and artefacts.

Thus, methods that would allow the reduction or the recognition of these elements in the sonified signal are investigated.

As concerns the intercritical activity, in the dataset such segments have a time duration <3s. Being very similar to epileptic activity they are not removed.

To remove the background noise, a patient-specific adaptive method is implemented. The method is based on the assumption that, in this case, the signal shows lower energy contribution as compared to an epileptic absence seizure, mainly as its characteristic frequencies are concerned.

TABLE 1 - Comparison of the proposed methods with the ONLINE method in [9] Method BACC F1score MCC FPR

> 69.0% ± 15.0%

> 73.0% ± 14.0%

> 72.0% ± 14.0%

> 71.0% ± 13.0%

and amplification. Only one channel is shown.

Figure 1 - Example of absence seizure sonification. Above: EEG signal (1 derivation); middle: sonification without postprocessing; below: sonification with post-processing

Figure 2 shows an example of artefact and the corresponding sonified signal, before (middle plot) and after (lower plot) the application of the techniques

An example of qualitative comparison is shown in Figure 1 where the sonified signal of an epileptic seizure and its EEG are presented. The upper plot shows the EEG, the middle plot shows the result of sonification without the post-processing procedure, the lower plot shows the result obtained after the application of the proposed additional post-processing

0.70 ± 14.0

0.73 ± 13.0

0.72 ± 13.0

0.72 ± 13.0

1.1% ± 1.0%

0.8% ± 0.9%

0.6% ± 0.7%

0.5 % ± 0.7%

> Figure 2 - Example of artefact sonification. Above: EEG signal (1 derivation); middle: sonification without postprocessing; below: sonification with post-processing

Finally, the results of the statistical tests described

0.53 ± 0.14 0.51±

IV. DISCUSSION

Table 1 shows an increase of the performance especially for the FPR parameter that is reduced by almost 50% as compared to the case without postprocessing. Also the F1score and MCC parameters increase with respect to the original ONLINE case. The decrease of the BACC parameter is an indication that some seizure windows (True Positive TP) were eliminated by the proposed method. Therefore, the choice of the parameter K must be made with caution to avoid losing useful information: indeed high values, such as K>3, could eliminate more seizures than

Regarding the amplification parameter α of the sonified signals, with and without post-processing, the results of Table 2 (case K = 2) show significant differences between TP and FP (False Positive) rates. Therefore, both the proposed techniques could alter the signal complexity, the TP Sample Entropy values decreasing more than the original ones, but less than the FP SE values. Possible reasons for this behaviour will be investigated in future developments of the

0.34 ± 0.18 0.14 ± 0.19

SE(2,1) – False positive

0.15

0.16

p-value Mann Whitney

3e^(-10)

1e^(-25)

0.04

TABLE 2 - Statistical test results. Sample Entropy SE(m=2, r=1) case K=2.

in the previous section are presented in Table 2.

True Positive

With α 0.35 ± 0.17 0.26 ±

Method SE(2,1) –

artefacts. This point is under study.

Frassineti et al.

With α and postprocessing

[9]

methods.

Frassineti et al. [9]

K=1 88.0% ±

K=2 86.0% ±

K=3 85.0% ±

described above.

89.0% ± 6.0%

6.0%

7.0%

8.0%

The proposed procedure is as follows. First, N windows (N = 100) with a duration of 2s each are selected from the signal. For each window the local maxima (Vlocal) of the rectified signal used for sonification are identified with a minimum distance between peaks given by 0.25\*fs and their average value is calculated. Once all the windows and their average values are obtained (Vmean\_local), a baseline is created for each patient given by the global mean value (Vmean\_global).

From the window N+1 on, the windows such that Vmean\_local ≤ Vmean\_global + K\*std(Vlocal [1:N]) are excluded.

The evaluation of the artefacts is made directly on the sonified signal. Considering that the energy trend of the artefacts should be discontinuous with respect to an absence seizure (always around 3Hz), an amplification term α is introduced in the sonification equation [9]:

$$\text{sound}\_{\text{abs}} = a \sum\_{\text{i}=\text{l}:\text{N}} \text{E}\_{\text{sound\\_i}} \sin(2\pi \text{f}\_{\text{osc}} \text{t})\tag{2}$$

where α is the square of the value of the peaks normalized with respect to their average and standard deviation and repeated for the minimum duration assigned to each peak. Quadratic amplification was chosen as a compromise between a linear amplification (difference between amplitude values difficult to detect) and an exponential one (excessive reduction of the sonified signal even with minimal differences in amplitude).

Therefore a sonified signal is obtained with periods of silence or discontinuous sound corresponding to artefacts, whereas for typical absences the amplitude of the signal is approximately constant.

Finally, likewise in [13], statistical tests are carried out to establish whether sonified signals of absence seizures are statistically different from false positives. In this work, the discriminant is the value of the Sample Entropy (SE) [15] in each time window of the sonified signal of length equal to 1s. The Mann Whitney test with significance level 0.05 is applied. For the Sample Entropy test, the embedding dimension m is set equal to 2 and the tolerance value r is set equal to 1.

### III. RESULTS

The performance is evaluated with the same metrics as in [9]: Balanced Accuracy (BACC); F1score; Matthews Correlation Coefficients (MCC); False Positive Rate (FPR). The comparison between the methods is presented in Table 1.

TABLE 1 - Comparison of the proposed methods with the ONLINE method in [9]

positives not discarded by the automatic recognition

absence seizure, mainly as its characteristic frequencies

The proposed procedure is as follows. First, N windows (N = 100) with a duration of 2s each are selected from the signal. For each window the local maxima (Vlocal) of the rectified signal used for sonification are identified with a minimum distance between peaks given by 0.25\*fs and their average value is calculated. Once all the windows and their average values are obtained (Vmean\_local), a baseline is created for each patient given by the global mean value

From the window N+1 on, the windows such that Vmean\_local ≤ Vmean\_global + K\*std(Vlocal [1:N]) are excluded. The evaluation of the artefacts is made directly on the sonified signal. Considering that the energy trend of the artefacts should be discontinuous with respect to an absence seizure (always around 3Hz), an amplification term α is introduced in the sonification

soundabs= α ∑i=1:N Esound\_i sin(2πfosct) (2)

where α is the square of the value of the peaks normalized with respect to their average and standard deviation and repeated for the minimum duration assigned to each peak. Quadratic amplification was chosen as a compromise between a linear amplification (difference between amplitude values difficult to detect) and an exponential one (excessive reduction of the sonified signal even with minimal differences in

Therefore a sonified signal is obtained with periods of silence or discontinuous sound corresponding to artefacts, whereas for typical absences the amplitude of

Finally, likewise in [13], statistical tests are carried out to establish whether sonified signals of absence seizures are statistically different from false positives. In this work, the discriminant is the value of the Sample Entropy (SE) [15] in each time window of the sonified signal of length equal to 1s. The Mann Whitney test with significance level 0.05 is applied. For the Sample Entropy test, the embedding dimension m is set equal to 2 and the tolerance value r is set equal

III. RESULTS

The performance is evaluated with the same metrics as in [9]: Balanced Accuracy (BACC); F1score; Matthews Correlation Coefficients (MCC); False Positive Rate (FPR). The comparison between the

the signal is approximately constant.

methods is presented in Table 1.

are concerned.

(Vmean\_global).

equation [9]:

amplitude).

to 1.

II. METHODS

To compare the performance of the proposed methods, the same EEG recordings used as test set in [9] and the same automatic recognition algorithms are considered here. The test set consists of 24 recordings of 22 children (7 males, 15 females, and age range 4 – 18 years) with absence seizures evaluated at the Paediatric Neurology Unit, Children's Hospital A. Meyer, Firenze, Italy. The duration of the signals is 47 ± 10 min with sampling frequency fs=256Hz. Written informed consent was obtained from all patients or

The proposed methods are implemented under MATLAB software (version 2017b [14]) installed on a Hp Pavillion 15 notebook (OS Windows 10, 64 bit) Intel Core i7-5500U processor, CPU 2.40 GHz, RAM

In [9] the automatic recognition gave 1% average percentage of false positives (FPR metric, ONLINE method) in the sonified signal. Here possible techniques that could further reduce false positives in

The sonification procedure proposed in [9] is based

Eq. (1) is described as follows. If the automatic recognition step detects three (or more) consecutive absences (ABS), the first 2 consecutive seconds will produce 2 beeps (beep1 and beep2) as pre-alarms. This sound represents a first discrimination between a

As described in detail in [9], the following seconds classified as ABS produce the soundabs. This is the sonified signal of the cumulated sum of the 6th levels of decomposition of the EEG signals obtained applying

False positives are grouped into: background without intercritical activity, intercritical activity or, more generally, elements of electrophysiological

Thus, methods that would allow the reduction or the recognition of these elements in the sonified signal

As concerns the intercritical activity, in the dataset such segments have a time duration <3s. Being very similar to epileptic activity they are not removed.

To remove the background noise, a patient-specific adaptive method is implemented. The method is based on the assumption that, in this case, the signal shows lower energy contribution as compared to an epileptic

[ABS – ABS – ABS] → [beep1beep2 → soundabs] (1)

the sonified signal are investigated.

possible absence and a false positive.

the Stationary Wavelet Transform.

interest of duration <2s, and artefacts.

are investigated.

on the following relationship:

process and the true epileptic seizures.

their guardians.

16 Gb.

An example of qualitative comparison is shown in Figure 1 where the sonified signal of an epileptic seizure and its EEG are presented. The upper plot shows the EEG, the middle plot shows the result of sonification without the post-processing procedure, the lower plot shows the result obtained after the application of the proposed additional post-processing and amplification. Only one channel is shown.

Figure 1 - Example of absence seizure sonification. Above: EEG signal (1 derivation); middle: sonification without postprocessing; below: sonification with post-processing

Figure 2 shows an example of artefact and the corresponding sonified signal, before (middle plot) and after (lower plot) the application of the techniques described above.

Figure 2 - Example of artefact sonification. Above: EEG signal (1 derivation); middle: sonification without postprocessing; below: sonification with post-processing

Finally, the results of the statistical tests described in the previous section are presented in Table 2.

### IV. DISCUSSION

Table 1 shows an increase of the performance especially for the FPR parameter that is reduced by almost 50% as compared to the case without postprocessing. Also the F1score and MCC parameters increase with respect to the original ONLINE case. The decrease of the BACC parameter is an indication that some seizure windows (True Positive TP) were eliminated by the proposed method. Therefore, the choice of the parameter K must be made with caution to avoid losing useful information: indeed high values, such as K>3, could eliminate more seizures than artefacts. This point is under study.

Regarding the amplification parameter α of the sonified signals, with and without post-processing, the results of Table 2 (case K = 2) show significant differences between TP and FP (False Positive) rates.

Therefore, both the proposed techniques could alter the signal complexity, the TP Sample Entropy values decreasing more than the original ones, but less than the FP SE values. Possible reasons for this behaviour will be investigated in future developments of the methods.

Figures 1 and 2 and the statistical results of Table 2 show the possibility of detecting different events in the sonified signal, at least in terms of sound intensity and its repeatability. However, given the large variety of possible artefacts in the EEG signal, the proposed method cannot capture all possible variants. In particular, those that have a rhythmic energetic trend with frequencies close to 3Hz would be difficult to differentiate from the typical absence seizures.

Given the low number of atypical epileptic absence seizures in the dataset (2 recordings), no statistical tests were carried out to assess differences with typical absences in the sonified signal.

### V. CONCLUSION

The application of post-processing and signal amplification techniques can lead to improvements both in terms of performance and qualitative recognition of events.

For a seizure detection system that applies sonification techniques the reduction of the FPR parameter seems relevant to reduce the amount of information to be analyzed.

The obtained statistical results allow performing validation experiments on the sonified signal [16]. The statistical evaluation of the sonified signal of atypical absences with respect to the typical ones will be one of the main future developments.

In conclusion, sonification techniques applied to EEG signals could introduce advantages for the realtime interpretation of information related to epileptic absence seizures. However, setting up sonification techniques for an easy removal of noise must be further exploited. If successful, they could help in reducing subjectivity and speed up the analysis.

### ACKNOWLEDGEMENT

This work was partially funded by Fondazione Cassa di Risparmio di Firenze, projects: 2016.0952 and 2015.0911.

### REFERENCES

[1] Epilepsy Atlas 2005 World Health Organization.

[2] E. Cesnik et al. "Incidence of epilepsy in Ferrara, Italy" Springer, Neurol Sci (2013) 34:2167-2172 doi 10.1007/s10072-013-1442-5.

[3] https://www.ilae.org/ last access: 10/09/2019

[4] R.S. Fisher et al. "Epileptic seizure and epilepsy: definitions proposed by the Internation League against Epilepsy (ILAE) and the International Bureau for Epilepsy (IBE)" Epilepsia 2005:46:470-472.

[5] R.S. Fischer et al. "Operational classification of seizure types by the International League Against Epilepsy: Position Paper of the ILAE Commission for Classification and Terminology." Epilepsia. 2017 Apr;58(4):522-530. doi: 10.1111/epi.13670.

[6] C. E. Stafstrom et al. "Seizures and Epilepsy: An Overview for Neuroscientists" Cold Spring Harb Perspect Med. 2015 Jun 1;5(6) doi: 10.1101/cshperspect.a022426.

[7] M. E. Saab and J. Gotman, "A system to detect the onset of epileptic seizures in scalp EEG," Clin. Neurophysiol., vol. 116, no. 2. pp. 427–442, Feb. 2005. [8] Vidyaratne, Lasitha S., Iftekharuddin, Khan M., 2017. "Real-time epileptic seizure detection using EEG". IEEE Trans. Neural Syst. Rehabil. Eng. 25 (11), 2146 –2156.

*Abstract:* **BioVoice is a user-friendly software for the acoustical analysis of the human voice. Here we introduce the last version of this software tool. Results of the acoustical analysis of newborn cry signals are described and discussed. Furthermore, results of the acoustical analysis of 2 adults (1 male and 1 female) and 1 child emitting sustained vowels**

Maria Sole Morelli<sup>1</sup>

*A. Biovoice: software description*

voice analysis.

, Silvia Orlandi<sup>2</sup> Claudia Manfredi <sup>1</sup>

**BIOVOICE: A MULTIPURPOSE TOOL FOR VOICE ANALYSIS**

<sup>1</sup> Department of Information Engineering (DINFO), Università degli Studi di Firenze, Firenze, Italy <sup>2</sup> Bloorview Research Institute, Holland Bloorview Kids Rehabilitation Hospital, Toronto, ON, Canada

<EMAIL_ADDRESS><EMAIL_ADDRESS><EMAIL_ADDRESS>
analysed.

results.

must be selected (Fig.2).

In Fig. 1, the main interface of BioVoice is shown. The user has to follow few mandatory steps to perform

First, the user selects and uploads at least one audio file. Indeed, it is possible to upload at the same time several files of any time duration and concerning different age range, gender and kind of voice emission. The file selection is allowed from any folder on the computer or HD or USB key. Only wav. files can be

Before starting the analysis, the user has to specify the settings of the audio file(s). Specifically, age (newborn, child, adult), gender (male or female), and kind of vocal emission (voiced, singing, speech, cry)

When the analysis starts, BioVoice first performs the selection of voiced/unvoiced (V/UV) audio segments [10]. Then, all the parameters of interests are extracted from each voiced segment. Specifically, in time domain, information about the number and length of voiced segments, length of silence segments and percentage of voiced segments are extracted and saved in an excel table. A picture shows the V/UV selection. In the frequency domain, fundamental frequency (F0), formant frequencies (F1, F2, F3), noise level (Normalized Noise Energy) and jitter are estimated. For F0 and for each formant, the mean, median, standard deviation, maximum and minimum values are calculated and saved in excel tables. Furthermore, differently from other automatic software tools, BioVoice computes the melodic shape of F0, identifying up to 12 melodic shapes (rising, falling, symmetric, plateau, low-up, up-low, double, frequency step, complex, unstructured, not a cry, other) [11].It is also possible to perform a perceptual melodic analysis, looking at each melodic shape of F0 and classifying them manually. Some other options are available. Indeed, a selected audio file can be listened to, and new audio files can be saved using a connected external microphone. For newborn and child, it is also possible to perform the perceptual analysis of the melodic shape and compare it with the automatic

*Keywords:* **BioVoice, acoustic analysis, software tool, voice parameters, infant cry, fundamental**

I. INTRODUCTION

Speech and vocal productions are characterized by acoustic waves generated by physiological processes that involve the central nervous system, the respiratory

The acoustical analysis of the human voice is considered clinically relevant in assessing the health state of the phonatory apparatus and detecting possible neurological disorders [1, 2]. Main clinical applications concern screening, diagnostic support and evaluation of the effectiveness of treatments [3]. To date, few automated methods have been developed for voice analysis, the most used being PRAAT [4]. Another one is the LENA system, that investigates both healthy child recordings and child populations with language

BioVoice, a user-friendly software tool for voice analysis, was developed at the Biomedical Engineering Lab, Firenze University [6–9]. It allows recording the human voice from the newborn to the elder and performing both time and frequency analysis, estimating more than 20 acoustical parameters. Here, a short description of the software is proposed. Furthermore, some results on voice analysis of newborn cries, child sustained vowels and adult

II. METHODS

**frequency, formant frequencies.**

system and the phonatory apparatus.

**are shown.**

disorders [5].

sustained vowels are shown.

[9] Frassineti et al. "Automatic detection and sonification of nonmotor generalized onset epileptic seizures: Preliminary results" Brain Research 1721 (2019) 146341.

[10] Kramer, G., et al., 1999. "Sonification report: status of the field and research agenda." Tech. Rep., Int. Commun. Auditory Display

[11] S. Schwarz et al. "A psychoacoustic sound design for pulse oximetry" 25th International Conference on Auditory Display (ICAD 2019) 23-27 June 2019 https://doi.org/10.21785/icad2019.024

[12] G.I. Mihalas et al. "Can Sonification Become a Useful Tool for Medical Data Representation?" MEDINFO 2017: Precision Healthcare through Informatics A.V. Gundlapalli et al. (Eds.) 2017 International Medical Informatics Association (IMIA) and IOS Press. doi:10.3233/978-1-61499-830-3-526

[13] F.B. Vialatte et al. "Audio representations of multi-channel EEG: a new tool for diagnosis of brain disorders" Am J Neurodegener Dis 2012; 1(3):292- 304.

[14] MATLAB and Statistics and Machine Learning Toolbox Release 2017b. The MathWorks, Inc., Natick, Massachusetts, United States.

[15] Richaman JS, Moorman JR. "Physiological timeseries analysis using approximate entropy and sample entropy." Am J Physiol Heart Circ Physiol 2000; 278: 2039-2049.

[16] Bonebright, T.L., 2011. "Evaluation of Auditory Display". In: Hermann, Thomas, Hunt, Andy, Neuhoff, John G. (Eds.), The Sonification Handbook. Logos Verlag, Berlin, Germany, pp. 111 –141.

### **BIOVOICE: A MULTIPURPOSE TOOL FOR VOICE ANALYSIS**

Maria Sole Morelli<sup>1</sup> , Silvia Orlandi<sup>2</sup> Claudia Manfredi <sup>1</sup>

<sup>1</sup> Department of Information Engineering (DINFO), Università degli Studi di Firenze, Firenze, Italy <sup>2</sup> Bloorview Research Institute, Holland Bloorview Kids Rehabilitation Hospital, Toronto, ON, Canada

<EMAIL_ADDRESS><EMAIL_ADDRESS><EMAIL_ADDRESS>
*Abstract:* **BioVoice is a user-friendly software for the acoustical analysis of the human voice. Here we introduce the last version of this software tool.**

Figures 1 and 2 and the statistical results of Table 2 show the possibility of detecting different events in the sonified signal, at least in terms of sound intensity and its repeatability. However, given the large variety of possible artefacts in the EEG signal, the proposed method cannot capture all possible variants. In particular, those that have a rhythmic energetic trend with frequencies close to 3Hz would be difficult to Epilepsy (ILAE) and the International Bureau for

[5] R.S. Fischer et al. "Operational classification of seizure types by the International League Against Epilepsy: Position Paper of the ILAE Commission for Classification and Terminology." Epilepsia. 2017

[6] C. E. Stafstrom et al. "Seizures and Epilepsy: An Overview for Neuroscientists" Cold Spring Harb Perspect Med. 2015 Jun 1;5(6) doi:

[7] M. E. Saab and J. Gotman, "A system to detect the onset of epileptic seizures in scalp EEG," Clin. Neurophysiol., vol. 116, no. 2. pp. 427–442, Feb. 2005. [8] Vidyaratne, Lasitha S., Iftekharuddin, Khan M., 2017. "Real-time epileptic seizure detection using EEG". IEEE Trans. Neural Syst. Rehabil. Eng. 25 (11),

[9] Frassineti et al. "Automatic detection and sonification of nonmotor generalized onset epileptic seizures: Preliminary results" Brain Research 1721

[10] Kramer, G., et al., 1999. "Sonification report: status of the field and research agenda." Tech. Rep.,

[11] S. Schwarz et al. "A psychoacoustic sound design for pulse oximetry" 25th International Conference on Auditory Display (ICAD 2019) 23-27 June 2019

[12] G.I. Mihalas et al. "Can Sonification Become a Useful Tool for Medical Data Representation?" MEDINFO 2017: Precision Healthcare through Informatics A.V. Gundlapalli et al. (Eds.) 2017 International Medical Informatics Association (IMIA) and IOS Press. doi:10.3233/978-1-61499-830-3-526 [13] F.B. Vialatte et al. "Audio representations of multi-channel EEG: a new tool for diagnosis of brain disorders" Am J Neurodegener Dis 2012; 1(3):292-

[14] MATLAB and Statistics and Machine Learning Toolbox Release 2017b. The MathWorks, Inc., Natick,

[15] Richaman JS, Moorman JR. "Physiological timeseries analysis using approximate entropy and sample entropy." Am J Physiol Heart Circ Physiol

[16] Bonebright, T.L., 2011. "Evaluation of Auditory Display". In: Hermann, Thomas, Hunt, Andy, Neuhoff, John G. (Eds.), The Sonification Handbook. Logos

Verlag, Berlin, Germany, pp. 111 –141.

Epilepsy (IBE)" Epilepsia 2005:46:470-472.

Apr;58(4):522-530. doi: 10.1111/epi.13670.

10.1101/cshperspect.a022426.

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Massachusetts, United States.

2000; 278: 2039-2049.

https://doi.org/10.21785/icad2019.024

2146 –2156.

(2019) 146341.

304.

Given the low number of atypical epileptic absence seizures in the dataset (2 recordings), no statistical tests were carried out to assess differences with typical

V. CONCLUSION

The application of post-processing and signal amplification techniques can lead to improvements both in terms of performance and qualitative

For a seizure detection system that applies sonification techniques the reduction of the FPR parameter seems relevant to reduce the amount of

The obtained statistical results allow performing validation experiments on the sonified signal [16]. The statistical evaluation of the sonified signal of atypical absences with respect to the typical ones will be one of

In conclusion, sonification techniques applied to EEG signals could introduce advantages for the realtime interpretation of information related to epileptic absence seizures. However, setting up sonification techniques for an easy removal of noise must be further exploited. If successful, they could help in reducing

ACKNOWLEDGEMENT

This work was partially funded by Fondazione Cassa di Risparmio di Firenze, projects: 2016.0952 and

REFERENCES [1] Epilepsy Atlas 2005 World Health Organization. [2] E. Cesnik et al. "Incidence of epilepsy in Ferrara, Italy" Springer, Neurol Sci (2013) 34:2167-2172 doi

[3] https://www.ilae.org/ last access: 10/09/2019 [4] R.S. Fisher et al. "Epileptic seizure and epilepsy: definitions proposed by the Internation League against

differentiate from the typical absence seizures.

absences in the sonified signal.

recognition of events.

information to be analyzed.

the main future developments.

subjectivity and speed up the analysis.

10.1007/s10072-013-1442-5.

2015.0911.

**Results of the acoustical analysis of newborn cry signals are described and discussed. Furthermore, results of the acoustical analysis of 2 adults (1 male and 1 female) and 1 child emitting sustained vowels are shown.**

*Keywords:* **BioVoice, acoustic analysis, software tool, voice parameters, infant cry, fundamental frequency, formant frequencies.**

### I. INTRODUCTION

Speech and vocal productions are characterized by acoustic waves generated by physiological processes that involve the central nervous system, the respiratory system and the phonatory apparatus.

The acoustical analysis of the human voice is considered clinically relevant in assessing the health state of the phonatory apparatus and detecting possible neurological disorders [1, 2]. Main clinical applications concern screening, diagnostic support and evaluation of the effectiveness of treatments [3]. To date, few automated methods have been developed for voice analysis, the most used being PRAAT [4]. Another one is the LENA system, that investigates both healthy child recordings and child populations with language disorders [5].

BioVoice, a user-friendly software tool for voice analysis, was developed at the Biomedical Engineering Lab, Firenze University [6–9]. It allows recording the human voice from the newborn to the elder and performing both time and frequency analysis, estimating more than 20 acoustical parameters. Here, a short description of the software is proposed. Furthermore, some results on voice analysis of newborn cries, child sustained vowels and adult sustained vowels are shown.

### II. METHODS

### *A. Biovoice: software description*

In Fig. 1, the main interface of BioVoice is shown. The user has to follow few mandatory steps to perform voice analysis.

First, the user selects and uploads at least one audio file. Indeed, it is possible to upload at the same time several files of any time duration and concerning different age range, gender and kind of voice emission. The file selection is allowed from any folder on the computer or HD or USB key. Only wav. files can be analysed.

Before starting the analysis, the user has to specify the settings of the audio file(s). Specifically, age (newborn, child, adult), gender (male or female), and kind of vocal emission (voiced, singing, speech, cry) must be selected (Fig.2).

When the analysis starts, BioVoice first performs the selection of voiced/unvoiced (V/UV) audio segments [10]. Then, all the parameters of interests are extracted from each voiced segment. Specifically, in time domain, information about the number and length of voiced segments, length of silence segments and percentage of voiced segments are extracted and saved in an excel table. A picture shows the V/UV selection. In the frequency domain, fundamental frequency (F0), formant frequencies (F1, F2, F3), noise level (Normalized Noise Energy) and jitter are estimated. For F0 and for each formant, the mean, median, standard deviation, maximum and minimum values are calculated and saved in excel tables. Furthermore, differently from other automatic software tools, BioVoice computes the melodic shape of F0, identifying up to 12 melodic shapes (rising, falling, symmetric, plateau, low-up, up-low, double, frequency step, complex, unstructured, not a cry, other) [11].It is also possible to perform a perceptual melodic analysis, looking at each melodic shape of F0 and classifying them manually. Some other options are available. Indeed, a selected audio file can be listened to, and new audio files can be saved using a connected external microphone. For newborn and child, it is also possible to perform the perceptual analysis of the melodic shape and compare it with the automatic results.

Claudia Manfredi (edited by), *Models and Analysis of Vocal Emissions for Biomedical Applications : 11th International Workshop, December, 17-19, 2019*, © 2019 FUP, CC BY 4.0 International, published by Firenze University Press (www.fupress.com), ISSN 2704-5846 (online), ISBN (online PDF) 978-88-6453-961-4

Fig.1 BioVoice user interface

Fig.2. BioVoice settings

At the end of the analysis, BioVoice results and pictures are saved in a specific folder created in the same directory of the audio file. Tables (excel) contain F0 and formants values and statistical information about the parameters. Colour figures (.jpeg) include: V/UV selection, F0 shape and spectrogram with formants values superimposed, for each detected voiced frame.

### *B. Data analysis*

To show some applications of BioVoice, we report the results of the analysis of four different recordings pertaining to different categories of human voice: a newborn cry (1st week of life) and sustained vowels of: a typically developing child (4 years old), an healthy adult male and an adult female (both 24 years old). Specifically, for the child and the adults only \a\, \i\ and \u\ vowels are considered here, as they allow building the vowel triangle, i.e. the plot of F2 vs F1 [12].

### III. RESULTS

In Fig.3, an example of the results of newborn cry analysis is shown. Specifically, in this recording six voiced frames (cry units) were detected with BioVoice.

with the first three formants superimposed is reported. In Fig.4, an example of sustained vowel for a child voice is reported (\a\), as well as its the melodic shape and spectrogram. Finally in Fig.5, an example concerning a male adult voice is shown (\a\). In Fig. 6, the vowel triangle of a child voice, of a male and a female adult voices are shown and compared to the normative values of male adult voice reported in [12].

Fig.4. Child voice (sustained \a\) a) V/UV selection; b) F0 shape; c) Spectrogram and formants for the first voiced unit.

F0= 237.5± 19.3 Hz Melodic shape: plateau

F1= 1286.3± 79.5 Hz F2= 1373.7 ± 76.8 Hz F3= 3862.9 ± 129.2 Hz

*a)*

*b)*

*c)*

shape analysis in newborn cry and child's voice, both

Fig.5. Adult male voice (sustained \a\) a) V/UV selection; b) F0 shape; c) Spectrogram and formants.

F0= 139.1 ± 1.6 Hz

F1= 687.1± 15.0 Hz F2= 1098.1 ± 41.9 Hz F3= 1851.6 ± 45.8 Hz

The reported results show some potential of this software. As for newborn cry analysis, BioVoice provides coherent values of F0 and formants in

Fig. 6. Vowel triangle: Comparison of children and adults results to normative values in [12].

automatic and perceptual.

*a)*

*b)*

*c)*

IV. DISCUSSION

We presented the last version of BioVoice, a software developed at the Firenze University for the acoustical analysis of human voice. This updated version is improved as of the interface layout is concerned, but most of all concerning the methodologies for parameter estimation. In addition to F0 and formants values, it allows performing melodic

Fig.3. Newborn cry. a) V/UV selection; b) F0 shape; c) Spectrogram and formants. First cry unit.

For each cry unit, F0, along with its mean and std and its melodic shape are computed. Also, the spectrogram

Fig.4. Child voice (sustained \a\) a) V/UV selection; b) F0 shape; c) Spectrogram and formants for the first voiced unit.

At the end of the analysis, BioVoice results and pictures are saved in a specific folder created in the same directory of the audio file. Tables (excel) contain F0 and formants values and statistical information about the parameters. Colour figures (.jpeg) include: V/UV selection, F0 shape and spectrogram with formants values superimposed, for each detected

Fig.2. BioVoice settings

To show some applications of BioVoice, we report the results of the analysis of four different recordings pertaining to different categories of human voice: a newborn cry (1st week of life) and sustained vowels of: a typically developing child (4 years old), an healthy adult male and an adult female (both 24 years old). Specifically, for the child and the adults only \a\, \i\ and \u\ vowels are considered here, as they allow building the vowel triangle, i.e. the plot of F2 vs F1

III. RESULTS

In Fig.3, an example of the results of newborn cry analysis is shown. Specifically, in this recording six voiced frames (cry units) were detected with BioVoice.

For each cry unit, F0, along with its mean and std and its melodic shape are computed. Also, the spectrogram

Fig.1 BioVoice user interface

*a)*

*b)*

*c)*

F0 mean = 448.6± 45.6 Hz Melodic shape: falling

F1 mean = 998.8± 91.4 Hz F2 mean = 2442.7 ± 88.8 Hz F3 mean= 5273.5 ± 148.5 Hz

Fig.3. Newborn cry. a) V/UV selection; b) F0 shape; c) Spectrogram and formants. First cry unit.

voiced frame.

*B. Data analysis*

[12].

with the first three formants superimposed is reported. In Fig.4, an example of sustained vowel for a child voice is reported (\a\), as well as its the melodic shape and spectrogram. Finally in Fig.5, an example concerning a male adult voice is shown (\a\). In Fig. 6, the vowel triangle of a child voice, of a male and a female adult voices are shown and compared to the normative values of male adult voice reported in [12].

### IV. DISCUSSION

We presented the last version of BioVoice, a software developed at the Firenze University for the acoustical analysis of human voice. This updated version is improved as of the interface layout is concerned, but most of all concerning the methodologies for parameter estimation. In addition to F0 and formants values, it allows performing melodic

shape analysis in newborn cry and child's voice, both automatic and perceptual.

Fig. 6. Vowel triangle: Comparison of children and adults results to normative values in [12].

The reported results show some potential of this software. As for newborn cry analysis, BioVoice provides coherent values of F0 and formants in different cry units. Concerning the melodic analysis, the software automatically detects the shape of F0, that can be compared to the perceptual one. As for child analysis, the comparison of its vowel triangle with normative values of adult male shows some differences. Indeed, as expected, children voice has higher values of F1 and F2 with respect to adults, the values of formants being related to the shape and size of the vocal tract: the larger the vocal tract cavities, the lower the resonance frequencies [12]. Concerning the adult's voice, the same consideration can be made. The vowel triangle of male voice is similar to the normative triangle reported in [12], while, as expected, for the female voice higher frequency values are shown, due to the smaller vocal tract cavities.

In the current version of BioVoice child and adult voice analysis is limited to voiced frames only. We are working towards including running speech analysis for both groups. Moreover, a revised version of the singing voice analysis is under development. In this case, two more formants, F4 and F5 must be estimated, as well as the so-called singer's formant and other acoustical parameters [13]. Finally, up to now the melodic shape is computed only for newborn cry and children voice, but it will be extended to the adult's voice too, as it might be related to the emotional state of the subject.

### V. CONCLUSION

A new version of BioVoice is presented. This software could be very useful for estimating several acoustical parameters, from the newborn to the elder. The software is very intuitive and easy to use also by a less expert user. The executable version is freely available upon request.

### REFERENCES

ENvironment analysis (LENA) system investigation of day long recordings in children: A literature review, *Journal of Communication Disorders*, Vol. 72, 77–85. doi:10.1016/J.JCOMDIS.2017.12.005

### **INDEX OF AUTHORS**

Agostini Tiziano 227 Aichinger Philipp 121, 125, 133, 137, 141 Álvarez A. 25, Angelakis Evangelos 51 Arora Siddharth 45

Barba C. 257 Becherini Cosimo 171 Biavati E. 81 Bouvet Anne 149, 153 Bruni Eleonora 81, 181 Bula Viteslav 205 Bulusu Sridhar 141 Burdumy Michael 179

different cry units. Concerning the melodic analysis, the software automatically detects the shape of F0, that can be compared to the perceptual one. As for child analysis, the comparison of its vowel triangle with normative values of adult male shows some differences. Indeed, as expected, children voice has higher values of F1 and F2 with respect to adults, the values of formants being related to the shape and size of the vocal tract: the larger the vocal tract cavities, the lower the resonance frequencies [12]. Concerning the adult's voice, the same consideration can be made. The vowel triangle of male voice is similar to the normative triangle reported in [12], while, as expected, for the female voice higher frequency values are shown, due

ENvironment analysis (LENA) system investigation of day long recordings in children: A literature review, *Journal of Communication Disorders*, Vol. 72, 77–85.

doi:10.1016/J.JCOMDIS.2017.12.005 [6] Orlandi, S.; Bocchi, L.; Donzelli, G.; Manfredi, C. (2012). Central blood oxygen saturation vs crying in preterm newborns, *Biomedical Signal Processing and Control*, Vol. 7, No. 1, 88–92.

doi:10.1016/J.BSPC.2011.07.003

doi:10.1016/J.BSPC.2008.11.006

[7] Manfredi, C.; Bocchi, L.; Cantarella, G. (2009). A multipurpose user-friendly tool for voice analysis: Application to pathological adult voices, *Biomedical Signal Processing and Control*, Vol. 4, No. 3, 212–220.

[8] Rruqja, N.; Dejonckere, P. H.; Cantarella, G.; Schoentgen, J.; Orlandi, S.; Barbagallo, S. D.; Manfredi, C. (2014). Testing software tools with synthesized deviant voices for medicolegal assessment of occupational dysphonia, *Biomedical Signal Processing and Control*, Vol. 13, 71–78. doi:10.1016/J.BSPC.2014.03.011 [9] Manfredi, C.; Bocchi, L.; Orlandi, S.; Calisti, M.; Spaccaterra, L.; Donzelli, G. P. (2008). Noninvasive distress evaluation in preterm newborn infants., *Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual Conference*, Vol. 2008, 2908–

2911. doi:10.1109/IEMBS.2008.4649811 [10] Orlandi, S.; Dejonckere, P. H.; Schoentgen, J.; Lebacq, J.; Rruqja, N.; Manfredi, C. (2013). Effective pre-processing of long term noisy audio recordings: An aid to clinical monitoring, *Biomedical Signal Processing and Control*, Vol.

[11] Manfredi, C.; Pieraccini, G.; Orlandi, S.; Viellevoye, R.; Torres-García, A.; Reyes-García, C. . (2019). Automated analysis of newborn cry: relationships between melodic shapes and native language, *Biomedical Signal Processing and*

[12] Deller, J. R.; Hansen, J. H. L.; Proakis, J. G. (2000). *Discrete-Time Processing of Speech*

[13] Manfredi, C., Barbagallo, D., Baracca, G., Orlandi, S., Bandini, A., Dejonckere, P.H. (2015) Automatic Assessment of Acoustic Parameters of the Singing Voice: Application to Professional Western Operatic and Jazz Singers, *Journal of*

*Voice*, Vol. 29, No. 4, 517.e1-517.e9

8, No. 6, 799–810

*Control*, Vol. (In print)

*Signals*

In the current version of BioVoice child and adult voice analysis is limited to voiced frames only. We are working towards including running speech analysis for both groups. Moreover, a revised version of the singing voice analysis is under development. In this case, two more formants, F4 and F5 must be estimated, as well as the so-called singer's formant and other acoustical parameters [13]. Finally, up to now the melodic shape is computed only for newborn cry and children voice, but it will be extended to the adult's voice too, as it might be related to the emotional state of the subject.

V. CONCLUSION

A new version of BioVoice is presented. This software could be very useful for estimating several acoustical parameters, from the newborn to the elder. The software is very intuitive and easy to use also by a less expert user. The executable version is freely available

REFERENCES [1] Niedzielska, G. (2001). Acoustic analysis in the diagnosis of voice disorders in children, *International Journal of Pediatric Otorhinolaryngology*, Vol. 57, No. 3, 189–193.

[2] Niebudek-Bogusz, E.; Fiszer, M.; Kotylo, P.; Sliwinska-Kowalska, M. (2006). Diagnostic value of voice acoustic analysis in assessment of occupational voice pathologies in teachers, *Logopedics Phoniatrics Vocology*, Vol. 31, No. 3, 100–106. doi:10.1080/14015430500295756 [3] Laver, J.; Hiller, S.; Beck, J. M. (1992). Acoustic waveform perturbations and voice disorders, *Journal of Voice*, Vol. 6, No. 2, 115–126.

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[5] Ganek, H.; Eriks-Brophy, A. (2018). Language

to the smaller vocal tract cavities.

upon request.

Calcinoni Orietta 171 Campos-Roca Y. 103 Cantarella Giovanna 167, 185 Chukaeva Tatjana 85 Ciabatta Annaclara 167 Contesse Adrien 219 Coppola Walter 227

D'Amario Sara 59 Daffern Helena 55 De Arcas-Castro Guillermo 21 Dehak Najim 33 DeJonckere Philippe 129, 163, 167 Devaraj Vinod 133 Di Natale Valentina 167 Dlask Pavel 63 Doroginskaya E.S. 245 Draginskaya E.S. Drioli Carlo 137

Echternach Matthias 179 Evain Solene 219 Evdokimova Vera 85, 237 Evgrafova Karina 85

Franke Ingolf 175 Frassineti L. 257 Frič Marek 63, 67 Fussi Franco 81, 181

Gálvez-García Gerardo 21 Ganapathy Sriram 223 Georgaki Anastasia 51 Godino-Llorente Juan Ignacio 33 Gómez A. 21, 25

Gómez P. 25 Gómez-García Jorge Andrés 33 Gómez-Rodellar Andrés 21 Gómez-Vilda Pedro 21 Gonet Wiktor 233 Greco Alberto 93 Grenez F. 37 Guerrini R. 257 Guhschina Y.S. 245 Gully Amelia 55

Hampala Vít 209 Hemmerling Daria 29 Henrich Bernardoni Nathalie 219 Hermansky Hynek 223 Hernández-del-Toro C. Tonatiuh 249 Horáček Jaromír 205, 213 Hortis-Dzierzbicka Maria 233 Howard David M. 59 Hruška Viktor 63

Israelsen Niels Møller 195

Jønsson Anders Overgård 195

Kacha A. 37 Kamanina N.A. 245 Kastyro I.V. 241, 245 Kim Y-J. 71, 89 Kovalenko A.N. 241, 245 Krenn Matthias 189 Kumar S. Pravin 141

Laukkanen Anne-Maria 205, 213 Lebacq J. 129 Lecouteux Benjamin 219 Lehoux Hugo 209 Leonhard Matthias 189 Lucero Jorge C. 145

Madruga M. 103 Manfredi Claudia XIII, 167, 253, 257, 261 Marceglia Sara 227 Martínez R. 25 Marzi Claudia 93 Mayr Winfried 189 Melani F. 257 Morales-Vargas E. 253 Morelli Maria Sole 261 Moro-Velázquez Laureano 33

Claudia Manfredi (edited by), *Models and Analysis of Vocal Emissions for Biomedical Applications : 11th International Workshop, December, 17-19, 2019*, © 2019 FUP, CC BY 4.0 International, published by Firenze University Press (www.fupress.com), ISSN 2704-5846 (online), ISBN (online PDF) 978-88-6453-961-4

Orlandi Silvia 261 Palacios D. 25 Palacios-Alonso Daniel 21 Pavani F. 97 Pedersen Mette 195 Pelorson Xavier 145, 149, 153 Peregrina-Barreto H. 253 Pérez C.J. 103 Pinchaud Antoine 219 Piras F. 257 Podzimková Iva 67 Prenassi Marco 227 Pützer Manfred 111 Patel Rita 107

Radolf Vojtěch 205, 213 Ramponi Giovanni 227 Reyes-García Carlos A. 249, 253 Richter Bernhard 179 Rosa E. 81

Schlömicher-Thier Josef 175 Schneider-Stickler Berit 189 Schoentgen J. 37 Schwab Didier 219 Scilingo Enzo Pasquale 93 Sidtis John J. 41, 71, 89

Skopich A. Skrelin Pavel 85 Spahn Claudia 179 Stoffels H. 163 Sundberg Johan 157 Svec Jan G. 141, 209 Sztaho David 29

Ternström Sten 107 Tokuda Isao 149 Torshin V.I. 241, 245 Traser Louisa 179 Tropper Hannes 175 Tsanas Athanasios 25, 45, 201

Van Hirtum Annemie 149, 153 Van Lancker Sidtis Diana 17, 41, 71, 89 Vanello Nicola 93 Vurma Allan 75

Wokurek Wolfgang 111,

Xi Chen 17

Yang Seung-yun 115

Zakharchenko E.A. 237 Zeloni G. 97 Zomkowska Edyta 233


*Article* **Seed Micromorphology,** *In Vitro* **Germination, and Early-Stage Seedling Morphological Traits of** *Cattleya purpurata* **(Lindl. & Paxton) Van den Berg**

**Miriam Bazzicalupo 1,\*, Jacopo Calevo 2,\*, Martino Adamo 2,3, Annalisa Giovannini 4, Andrea Copetta 4 and Laura Cornara 1**

**Abstract:** In the context of a symbiotic plant-fungus interaction study concerning *Cattleya purpurata*, we focused on some aspects of seed morphology and biology, and the early stages of seedling development. Seed morphology was characterized using light and scanning electron microscopy. In vitro seed germination capability was evaluated, comparing symbiotic and asymbiotic methods. The morphology of the seeds was overall comparable to that of other congeneric species, showing classical adaptations related to the aerodynamic properties and to the wettability of seeds, but calcium oxalate druses were identified inside the suspensor cells. Asymbiotic seed germination was successful in all tested media (17.1–46.5%) but was higher on 1/2 Murashige & Skoog. During symbiotic interaction with the fungal strain MUT4178 (*Tulasnella calospora*), germination rate was significantly lower than that obtained with the best three asymbiotic media, suggesting a low fungal compatibility. Seedling morphology was in line with other taxa from the same genus, showing typical characteristics of epiphytic species. Our observations, in particular, highlighted the presence of stomata with C-shaped guard cells in the leaves, rarely found in Cattleyas (where usually they are reniform), and confirm the presence of tilosomes in the roots. Idioblasts containing raphides were observed in both roots and leaves.

**Keywords:** crystals; microscopy; mycorrhizal fungi; orchids; suspensor

#### **1. Introduction**

Orchidaceae, with about 28,000 species and new ones being discovered every year, is one of the largest families of flowering plants [1]. The great majority of species is native to tropical or sub-tropical areas, but the family can be considered cosmopolitan, being spread over the five continents, with a distribution range that extends from the Arctic Circle to sub-Antarctic islands [2]. Orchids have interesting and often strict interactions with specialized pollinators and mycorrhizal fungi [3–5], and are at risk of extinction by intrinsic and extrinsic factors linked with habitat degradation [6], including weed invasion, grazing, altered hydrology, and altered fire regimes.

Among the Epidendroideae subfamily, the subtribe Laeliinae is strictly neotropical and comprises about 50 genera with about 1500 species [7,8]; it is one of the largest subtribes in the family after Pleurothallidinae and Oncidiinae [9]. Due to its morphological variability and taxonomic divergence [9], Laeliinae has been the subject of great taxonomic reviews

**Citation:** Bazzicalupo, M.; Calevo, J.; Adamo, M.; Giovannini, A.; Copetta, A.; Cornara, L. Seed Micromorphology, *In Vitro* Germination, and Early-Stage Seedling Morphological Traits of *Cattleya purpurata* (Lindl. & Paxton) Van den Berg. *Horticulturae* **2021**, *7*, 480. https://doi.org/10.3390/ horticulturae7110480

Academic Editor: Sergio Ruffo Roberto

Received: 11 October 2021 Accepted: 8 November 2021 Published: 10 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

over the last few decades; in this subtribe, Dressler [7] included the genera *Cattleya* Lindl., *Laelia* Lindl., *Schomburgkia* Lindl., and *Sophronitis* Lindl. Van den Berg et al. [9,10], thanks to a phylogenetic analysis on ITS regions and considering the hybridization capability within Laeliinae, supported the inclusion of *Schomburgkia* and *Sophronitis* species in *Laelia* and *Cattleya*, respectively. For the above-mentioned reasons, Brazilian *Laelia* species, previously referred to as *Hadrolaelia* [11], have also been included in the genus *Cattleya* [12].

*Cattleya purpurata* (Lindl. & Paxton) Van den Berg (syn. *Laelia purpurata*) is a Brazilian orchid species that grows widely in the country and has been ranked as endangered (EN) by IUCN (International Union for Conservation of Nature) in the State of Rio Grande do Sul [13], due to its over-collection for commercial purpose [14]. The species has been the focus of many studies due to its high economical value; it is considered one of the flag flowers of Brazil and nowadays it is in collections all over the World, with several cultivars and varieties [15]. As recently reported by Caballero-Villalobos et al. [16], the species is nectarless and self-compatible, although it is pollinator-dependent. Some morphological investigations on the vegetative portions of this species have been carried out by Silva Júnior et al. [17] and Gallo et al. [18]. The former studied leaf characteristics and chlorophyll content of this species after the applications of different urea concentrations during *in vitro* experiments. Gallo et al. [18] performed an anatomical investigation on seed and protocorm from six species belonging to the genus *Cattleya*, *C. purpurata* included. Anatomical investigations provide important information to assess species identity, to better understand physiological processes, or to study phylogenetic relationships between taxa [19]. This information, which can be achieved considering both the vegetative and reproductive portions of a species, could also be used to improve knowledge and be applied in micropropagation techniques and conservation programs [18].

New propagation techniques have been proposed over the last decades to optimize asymbiotic and symbiotic seed germination, and to promote seedling acclimatization [15,20,21]. However, contrary to what is known about terrestrial species [22–24], few details are available concerning mycorrhizal associations in tropical orchids, especially in Laeliinae occurring in different environments (i.e., [25,26]).

In this work, which has been carried out in the framework of a plant-fungus interaction study [27], our aim was to obtain more morphological information regarding *C. purpurata* seed traits and test seed germination via both asymbiotic and symbiotic techniques, in the latter case using a fungal strain of the well-known orchid mycorrhizal (OM) fungus *Tulasnella calospora.* Seedlings grown on different media and growth conditions were then characterized from an anatomical and micro-morphological point of view in order to collect further data concerning this stage of development and to improve propagation techniques to be used in scientific and conservation programs.

#### **2. Materials and Methods**

#### *2.1. Flower Hand Pollination and Seed Collection*

Capsules were obtained by hand pollinated plants of *Cattleya purpurata* cultivated by the Azienda Agricola Nardotto e Capello in Camporosso (Imperia, Italy), 43◦48 1800 N, 07◦37 4332 E. At the time of capsule maturation, indicated by a yellowish color, three capsules were collected, and seeds were stored at 4 ◦C in a paper envelop until use [28].

#### *2.2. Seed Sowing*

After a sterilization in 1% NaClO solution, seeds were rinsed three times in sterile dH2O for 5 min and then sowed on six different asymbiotic seed germination media: 1/2 strength of Murashige and Skoog [29], including vitamins (Duchefa) enriched with 50 mL/L of coconut water; Knudson C [30] orchid medium (Duchefa); Malmgren Modified medium [31]; M551 (Phytotechnology) enriched with 1mg/L BAP, which are commonly used for the propagation of epiphytic and terrestrial orchids [32]; and CG (CG0) and CG enriched with 50 mL/L (CG50) and 100 mL/L (CG100) almond milk, a new medium that showed to improve *in vitro* seedling development in both epiphytic and terrestrial

orchids [32]. All the media were supplemented with 2 g/L of activated charcoal and a total concentration of 10 g/L of sucrose; pH was adjusted to 5.8. Seeds were also sowed on an oat agar medium (OA), as described by Ercole et al. [33], with a mycorrhizal fungal strain of *Tulasnella calospora*, isolated at the University of Turin from *Serapias vomeracea* [34] and deposited in the mycological collection of the University of Turin (Mycotheca Universitatis Taurinensis, accession number MUT4178). Eight replicates for each medium were periodically checked under light microscope to assess germination percentage. The final germination percentage, recorded 70 days after seed sowing, was evaluated as the overall mean of single observations ± SE. Seedlings were then moved to jars with the same media for three months before recording morphological data.

#### *2.3. Microscopy*

#### 2.3.1. Plant Material

Nine months after sowing (after transferring seedlings on fresh media every three months), fully developed roots and leaves from each medium were sampled. Roots and leaves were cut about 1 cm from the apex and about half of their length, respectively. Plant material was prepared to perform micro-morphological investigations according to the following methods.

#### 2.3.2. Light Microscopy

Light microscopy (LM) analysis was carried out on seeds, seedling leaves, and roots. Seeds were directly mounted onto a microscope slide after being rinsed in 5% NaClO solution for 1 h, and were then observed with a Leica DM 2000 transmission-light microscope coupled with a computer-driven DFC 320 camera (Leica Microsystems). Seed and embryo length, length/width (L/W) ratio, number of cells along the entire seed longitudinal axis, and length and number of cells of the suspensor were recorded for 100 seeds. Seed descriptive terminology has been used according to Barthlott et al. [35].

Transversal hand-sections of fresh leaves and roots obtained by a razor blade were stained with both TBO (Toluidine Blue O), to discriminate cell wall structures, and acid Phloroglucinol, to highlight xylem and lignified structures in general. The seeds and transversal sections of leaf and roots were also examined under a polarizing filter. In addition, to localize suberin and lignin, transversal sections of roots were stained with Fluorol Yellow 088 and observed under UV light.

#### 2.3.3. Scanning Electron Microscopy

Seeds were incubated overnight in 100% ethanol, placed on aluminum stubs covered with double-sided carbon tape, and dried at room temperature, as performed by Calevo et al. [36]. Stubs were then sputter-coated with 10 nm gold particles and directly observed with a Vega3 Tescan LMU Scanning Electron Microscope (SEM) (Tescan USA Inc., Cranberry Twp, PA, USA) at an accelerating voltage of 20 kV, coupled to an X-ray Energy Dispersive System (EDS) Apollo XSD (Tescan USA Inc., Warrendale, PA, USA). A detailed assessment of seed ornamentation was carried out, considering the ornamentation of the periclinal walls and the type of anticlinal walls [37].

For SEM analysis of leaves and roots from in vitro grown seedlings, transversal sections of fresh material were fixed in FineFIX working solution (Milestone s.r.l., Bergamo, Italy) with 70% ethanol, and left overnight at 4 ◦C [38]. Sections were then dehydrated for 1 h through graded ethanol series from 70% to 100% at 60 min intervals, and finally dried using a Critical Point Dryer Processor (K850CPD 2M Strumenti S.r.l., Roma, Italy). Specimens were then mounted on stubs, as previously performed on seeds, to carry out micromorphological analyses.

#### *2.4. Data Analysis*

Germination data were statistically analyzed by ANOVA followed by Fisher's probable least-squares difference test, with a cut-off significance at *p* ≤ 0.001.

Embryo and seed volumes were calculated following the equations reported in [37]:

$$\text{Embryo volume (EV)} = 4/3 \text{ }\pi \text{ab}^2 \tag{1}$$

$$\text{Speed volume (SV)} = 2(\pi/3 \text{ r}^2 \text{h})\tag{2}$$

where a = 0.5 × embryo length (or major axis); b = 0.5 × embryo width (or minor axis); r = 0.5 × seed width (or minor axis); and h = 0.5 × seed length (or major axis).

The morphological data of seeds were analyzed using the R environment [39]. Correlations between seed and embryo volumes were analyzed by means of the cor.test function, using the Spearman's rank correlation coefficient rho [40,41]. Measures were expressed in μm ± SD.

#### **3. Results**

#### *3.1. Seed Germination*

*Cattleya purpurata* seeds germinated on all the tested media. The best medium for asymbiotic germination was 1/2 MS, with a germination percentage of 46.5 ± 6.4%. All the other asymbiotic media yielded lower germination percentages, ranging from 17.1 ± 2.0% (CG0) to 26.3 ± 4.3% (KC). The use of MUT4178 fungal strain of *T. calospora* resulted in a germination of 18.3 ± 4.0%, statistically comparable with all the variants of CG medium (Table 1).

**Table 1.** Germination mean percentages (± Standard Error) (*n* = 8) recorded 70 days after seed sowing of *Cattleya purpurata*. Data were analyzed by ANOVA followed by Fisher's probable leastsquares difference test with cut-off significance at *p* ≤ 0.001. Percentages with the same letters are not significantly different from each other.

#### *3.2. Morphology*

#### 3.2.1. Seed Morphology

*Cattleya purpurata* seeds, beige in color, are elongated (Figure 1a) and present a L/W ratio of 5.33 ± 0.62. They showed an average seed length (excluding the suspensor) of 800.47 ± 97.42 μm, an average width of 154.75 ± 22.37 μm, an embryo length of 331.59 ± 48.04 μm, and an embryo width of 127.22 ± 19.98 μm (Table 2). The comparison of seed and embryo volumes showed a significant positive relationship (R = 0.74, *p* < 0.001) (Supplementary Figure S1). The average suspensor length was 302.52 ± 64.53 μm; no significant relationship was found between suspensor length and both seed and embryo length and either between suspensor length and seed and embryo volumes.

**Figure 1.** Light microscopy images of *Cattleya purpurata* seed. (**a**) LM overview of NaClO-treated seed, in which embryo is well distinguishable within the seed coat; (**b**) detail of the embryo body-suspensor connection (arrow) composed of 2–3 elongated cells. (**c**) untreated seed: View of the insertion of the suspensor inside the micropyle. Spherical druses are present in each cell of the suspensor (arrow).

> **Table 2.** Means of seed morphological parameters (*n* = 100) and seed features of *Cattleya purpurata* (± Standard Deviation).

Clarifying treatment with NaOCl allowed us to verify that seed coat was monolayered and that there were no intercellular gaps. Concerning *testa*, more than five cells were present along the seed longitudinal axis. These cells were elongated; their periclinal cell walls presented verrucosities and granular epicuticular coverings, while their anticlinal walls were occasionally elevated. As shown in Figure 2a,b, the seed coat enveloped the embryo, which showed a relatively long suspensor. Two-three elongated cells connected the embryo body with the portion of suspensor extending beyond the micropyle (Figure 1b, arrow).

**Figure 2.** SEM micrograph of *Cattleya purpurata* seed. (**a**) overview of the seed showing the suspensor (arrow); (**b**) detailed view of the suspensor made of a 2–3 cells-thick layer; (**c**) cutaway of the suspensor highlighting the presence of druses (arrows) inside each cell. At higher magnification, the verrucose microrelief and the granular ornamentation of the seed coat cells are also visible; (**d**) particular of the druses (arrows).

The presence of spherical druses (6–8 nm in diameter) within the seed suspensor cells was recorded by both light (Figure 1a,c) and scanning electron microscopy (Figure 2c,d). SEM-EDS analysis of these crystals showed a high calcium peak, typical of the calcium oxalate spectrum (Supplementary Figure S2).

#### 3.2.2. Root Sections

Microscopical analyses highlighted that the structure of roots sampled from *C. purpurata* seedlings grown on different substrates was qualitatively similar, except for the increased presence of hairs in individuals from CG50 and CG100 media (not shown). Velamen was two to four cells wide (Figure 3a); these cells appeared large, polygonal shaped, and radially elongated, and showed helical-banded wall thickenings (Figure 3b,g).

Exodermal cells were moderately large, polygonal-shaped, and ∩-thickened (Figure 3b,d). Short thin-walled passage cells were scattered in the exoderm, near tilosomes of tufted type (Figure 3b, arrows) (according to Pridgeon et al. [42]). In the parenchymatous cortex, the presence of chloroplasts (Figure 3d) and raphides (Figure 3e) was recorded. Cortical cells varied in form and size: elements near to the endodermis were smaller than those of the middle layers (Figure 3a,d); some parenchymatous cells appeared more or less circular, while others were polygonal-shaped (Figure 3a,b,d). As confirmed by means of polarized light (not shown) and by Fluorol Yellow staining, some elements of the cortex presented suberized thickenings in cell walls (Figure 3c, arrows). Endodermis cells were square to tetragonal-shaped; passage cells were visible in correspondence of phloem clusters, while the other endodermis cells appeared O-thickened (Figure 3c). The pericycle presented thin-walled polygonal-shaped cells alternating with protoxylematic cell elements. This layer surrounded the central vascular cylinder characterized by a polyarch actinostele, which showed 10 xylem arches. In the root's central region, a parenchymal tissue composed by circular cells alternating with lignified elements was present (Figure 3c).

Roots of seedlings obtained from symbiotic cultures showed the presence of mycorrhizal hyphae on the external surface (Figure 3f). The beginning of hyphal coiling was also well visible on the wall thickening of the velamen cells (Figure 3g). Root features are summarized in Table 3.

**Root Characters** Hairs From few to numerous if roots belonged to plants grown in CG50 and CG 100 Velamen cells Moderately large, polygonal shaped, arranged in 2–3 layers. Helical thickenings in cell walls Exodermis cells Large, polygonal-shaped, arranged in monolayer. ∩-thickened. Cortex cells Circular to polygonal shaped. Variable size. Presence of chloroplasts. Some elements show thickenings in the cell walls. Endodermis cell walls square to polygonal shaped, O-thickened;

**Table 3.** General seedling's root and leaf anatomical characters of *Cattleya purpurata* observed on all tested media (unless otherwise indicated).

**Figure 3.** Root anatomy of *Cattleya purpurata* seedlings. Representative seedlings obtained on 1/2MS were selected for panels (**a**–**e**) and from OA medium for panels (**f**,**g**). (**a**) overview of the root's transversal section stained with Phloroglucinol-HCl. Velamen is composed by a 2–3 cells wide layer; exoderm cells appear large, polygonal, and ∩-thickened; cortex cells from middle layers are larger than those near to the exoderm and to the endoderm. The vascular cylinder is composed by a polyarch actynostele; (**b**) tufted tilosomes (arrows) observed in the endovelamen-exoderm region; (**c**) Fluorol Yellow staining: detail of the central vascular cylinder characterized by a polyarch actinostele. The central parenchymatic tissue is composed by circular cells alternating with lignified elements. Cortex elements showing suberized cell walls are also visible (arrows); (**d**) chloroplasts within cortex cells; (**e**) SEM photograph of bundledraphides (br) within a cell of cortical parenchyma; (**f**) SEM image of mycorrhizal hyphae in the first stages of colonization of a root obtained from symbiotic culture; (**g**) the beginning of hyphal coiling inside a velamen cell (arrow). Banded wall thickenings of velamen cells are also distinguishable.

#### 3.2.3. Leaf Surface and Transversal Sections

The media used for seed germination and seedling development did not lead to structural changes in leaves. Leaf surfaces were composed of mostly rectangular to hexagonal cells and showed a ridged, granular cuticle, 7.0 ± 1.3 μm thick in the abaxial surface and 10.0 ± 1.6 μm in the adaxial one (Figure 4a,b). Tetracytic stomata with C-shaped guard cells were present only in the lower epidermis; the stomatal aperture was parallel to the longitudinal axis of the leaf (Figure 4a). Mean stomatal length was 23.5 ± 2.9 μm, while mean stomatal width was 26.5 ± 2.6 μm. In transversal section, on both sides of the midvein, seven vascular bundles arranged in a row were visible (Figure 4c). The central midvein (Figure 4c,d), positioned near the lower surface, was surrounded by several hypodermal cell layers (not shown). In correspondence of the central midvein, the epidermis was bistratified. The leaf presented a dorsiventral mesophyll at the level of the central midvein and a homogeneous one towards the margins. Parenchyma cells gradually increased in diameter while approaching the central portion, where isodiametric and elongated thick-walled cells were present near the midvein, resembling a palisade parenchyma (Figure 4c). In many cases, we observed vessel elements developed transversely, which connected the small veins (Figure 4e). Vascular collateral bundles showed sclerenchyma caps at both xylem and phloem poles, but predominantly at the phloem one (Figure 4d,e). Little sclerenchyma fiber bundles were present as an abaxial row (not shown), while two clearly distinguishable bundles were positioned at each leaf margin: a little bundle in the margin tip and a larger one more internal (Figure 4c,f). Idioblasts containing raphides were interspersed in the mesophyll (Figure 4f, arrow). Leaf features are summarized in Table 3.

As highlighted by SEM analyses, some mesophyll cell walls were banded (Figure 4g).

**Figure 4.** Leaf anatomy of *Cattleya purpurata* seedlings. Representative seedlings obtained on M551 medium were selected for images. (**a**) SEM picture of the lower surface of the leaf, revealing hexagonal cells covered by a thick granular cuticle and the tetracytic stomatal apparatus with C-shaped guard cells; (**b**) transversal section: detail of the leaf's abaxial portion, in which stomata are visible. Cuticle is 6–9 μm thick; (**c**) transversal section stained with TBO. Seven vascular bundles arranged in a row are visible. The central midvein is positioned near the lower surface. A dorsiventral mesophyll is present at the level of the central midvein, while a homogeneous one is distinguishable towards the margins. Two sclerenchymatic bundles are present at each leaf tip; (**d**) View of the central midvein stained with HCl-phloroglucinol; (**e**) transversely developed vessel elements, connecting with a vascular bundle red-stained with HCl-phloroglucinol; (**f**) transversal section of the leaf stained with TBO. The two sclerenchymatic bundles are visible near the leaf margin; (**g**) SEM picture of the mesophyll in cross section: banded thickenings are distinguishable in some cells.

#### **4. Discussion**

As observed by Gallo et al. [18] and confirmed by our analyses, the seed coat of the studied species consists of only one cell layer. As reminded by the latter authors, the reduced and membranous seed coat of Laeliinae seeds and the air space between the tegument and the embryo is an adaptation related to the aerodynamic properties and to the wettability of seeds [37,43]. While a significant correlation has been found between seed and embryo length, suspensor length was not correlated to seed or embryo size, indicating that suspensor dimension does not influence seed development in *C. purpurata*.

Although Gallo et al. [18] already described the long multicellular suspensor in *C. purpurata* and other specimens belonging to the Laeliinae subtribe, the presence of calcium oxalate druses for each cell of the structure had not yet been reported. To the best of our knowledge, this study is the first reporting the occurrence of druses in the suspensor, or more generally, in orchid seeds. In fact, such crystal conglomerates, according to Prychid and Rudall [44], are commonly found in various dicotyledons tissues but are more rarely observed in monocotyledons; concerning family Orchidaceae, druses have been recorded only in vegetative portions of Epidendrae and Dendrobieae [45,46], Arethusae [46], and in the >23% of the Oncidiinae [47].

Lee et al. [48], studying the suspensor of *Paphiopedilum delenatii*, observed that there was no cuticular material in the cell walls of the structure; considering the morphological traits of the transfer cell, they finally hypothesized that the suspensor was responsible for nutrient uptake for the embryo. Later, the role of embryo suspensor in nutrition has been confirmed [49,50]. In addition, Volk et al. [51] demonstrated that the deposition of druses in idioblasts is a dynamic process and, when Ca availability is reduced, cells could dissolve the crystals metabolizing CaOx to non-toxic compounds. According to these authors and considering our observations, it is possible that the suspensor possesses storage potential and, while providing a channel for nutrients conduction to embryo, could store the excess of calcium coming from the environment or could receive it as a byproduct of seed metabolism. Calcium oxalate, on the other hand, has many functional roles also for fungi, such as metal detoxification or increasing plant susceptibility to fungal infection, acting as an electron donor in lignocellulose degradation [52] and in the reduction of certain metals [53]. Oxalate, indeed, plays a unique role in lignocellulose degradation by basidiomycetes, acting as a low molecular mass agent initiating decay and as a potential electron donor for ligninperoxidase catalyzed reduction [54–56]. This is particularly interesting because fungal colonization of orchid seeds by orchid mycorrhizal fungi (belonging to the Basidiomycota) occurs through the suspensor [57–60]. It is tempting to hypothesize that the druses have evolutionarily assumed the role of ready-to-use oxalate reserves available to the symbiont fungi to favor the degradation of the lignocellulose present in the seed coat [61] and their settlement as symbionts. It should be remembered that, in mycorrhizal fungi, the energy balance is very important [22] and even more so in orchid mycorrhizal fungi, where the debate in the scientific community is still ongoing [22,62,63]. Possibly supporting this hypothesis, Miura et al. [64] found that the presence of seed coat enhances seed germination with symbiotic fungi and protects the embryo against the attack of non-symbiotic fungi by restricting the invasion of their fungal hyphae. Indeed, the seed coat is a barrier against fungi that do not possess enzymes capable of degrading lignocellulose, making a sort of selection at the entrance.

However, as the druses deposition was recorded starting from the first two cells after the elongated pair and all along the suspensor (see Figure 3a,b), we cannot exclude that its detachment from the developing embryo in the early stages of germination might be a fast way to remove the excess of calcium oxalate. This hypothesis agrees to that discussed by Paiva [65], who concluded that the formation of CaOx crystals in portions that will be discarded allows the excretion of calcium, because plants lack excretory systems.

Germination was successful on all the media tested, but 1/2 MS was the best and is therefore suggested for *C. purpurata* propagation. Germination occurred also by using the fungal strain MUT4178 of *Tulasnella calospora* but with a lower percentage if compared to the above-mentioned asymbiotic medium. Almeida et al. [26] indicated Tulasnellaceae (including *T. calospora*) as the main possible fungal symbionts for this orchid group; however, our results, which report the first attempt of symbiotic germination for *C. purpurata* (to the best of our knowledge) may indicate that the fungal strain used, isolated from an European terrestrial orchid, might not be the most appropriate or the dominant symbiont for this species, even if we demonstrated its colonization of roots (see also Adamo et al. [27]).

The subsequent development of seedlings grown *in vitro* allowed characterization of their morphology. According to the literature, this is the first study providing morphological information regarding *in vitro* seedlings of *C. purpurata*, because Gallo et al. [18] worked on the same species but in earlier stages of protocorm development, while Silva Júnior et al. [17] compared the anatomy of roots and leaves from plants treated with different urea concentrations.

We observed idioblasts containing raphides in both root and leaf tissues, adding information regarding their presence in the early development stages of seedlings. The occurrence, distribution, and morphology of idioblasts containing raphides have been reported by various authors who studied the vegetative tissues of most of the analyzed orchid subtribes [66] (and references therein). Stern and Carlsward [67] carried out a comparative study concerning vegetative portions of members from Laeliinae, and found that crystalliferous idioblasts, circular in transverse section and saccate in the longitudinal one, are present ubiquitously in the mesophyll of all the examined taxa. In general, the role of raphides for plant tissues also needs to be clarified. As recently revised by Paiva [65], various hypotheses about the functions of raphides in plant tissues have been indeed formulated; they could help in regulating calcium levels [51] or constitute a CO2 source for photosynthesis in some species [68]. Tulyananda and Nilsen [69] stated that idioblasts spread in thin leaves of epiphytic species (in this case, *Rhododendron* sp.) act as buffering agents that significantly affect leaf-lamina water relations. As previously proposed by several authors and recently revised by Konno et al. [70], raphides could play a defensive role against herbivory.

Some of the anatomical characters observed in the roots and leaves of the studied species are typical to those of epiphytic orchid habit.

Root presented a 2–3 layered velamen, showing helical cell wall thickenings. Such a restricted number of velamen layers is characteristic of species belonging to relatively humid habitats [71]. Velamen avoids the loss of moisture from roots, speeds up water absorption, and provides protection against mechanical stresses and ultraviolet radiations [72,73]. On the other hand, helical thickenings in the velamen cells have the function to improve the stability and efficiency of this tissue for water absorption by the root and its retention in dry conditions in epiphytic orchids [74]. Passage cells constitute a channel for the selective transit of nutrients and water, and for the entrance of mychorrhizal fungi [71,75,76]. Tilosomes, branched lignified wall ingrowths on the internal periclinal wall of the endovelamen cells, are typically positioned near passage cells [77,78]. These structures, according to Kedrovski and Sajo [78], increase symplastic connections and improve water and solutes transport between external and internal environments in young tissues, while they steer solutes to passage cells in mature tissues. Stern and Carlsward [67] stated that there are no tilosomes in *Cattleya* but annotated their occurrence in other species that have been moved in this genus in times after their work, i.e., *Sophronitis sincorana* (now *C. sincorana*) and *Laelia anceps* (now *C. anceps*). Our observation confirms the fact that tilosomes are also present in *Cattleya* genus.

The monolayered exodermis with large elements with ∩-thickened cell walls is also typical of epiphytic orchids. This structure prevents transpiration and water loss from the cortex [71].

Taken together, the characteristics of exodermis, the number of velamen layers, the occurrence and orientation of thickenings in velamen cell walls, and the presence of tilosomes possess taxonomical value according to Porembsky and Barthlott [77]. To classify orchid roots, the latter authors indeed considered combinations of the aforementioned characters, proposing 12 different syndromes; the complex of features showed by our study species resembles that of *Vanda*-type.

A high presence of chloroplasts was recorded in the cortex. This aspect is well known for Laeliinae [67] and in general for epiphytic orchids; many species have evolved photosynthetic roots to increase photosynthetic areas and consequently carbon gain [72,79,80].

The variable shape and the presence of thickened birefringent cell walls of cortex elements agree with what has been reported for other Laeliinae members by Stern and Carlsward [67]. Additionally, both endodermis and pericycle cells show characteristics already described by the latter authors (similar in dimension and O-thickened); however, contrary to what has been observed in general for Laeliinae, we recognized O-thickenings in correspondence of xylem rays instead of phloem ones, and protoxylematic elements in the pericycle. Lignin deposition in other elements of the central cylinder and endodermal cell wall thickenings may play similar functions to those of velamen and exodermis (prevention of water loss) and constitute another adaptative trait to tropical habitat conditions [71].

The number of xylem arches (10) falls in the range of 7–24, reported for this group of orchids [67]. Gallo et al. [18] recorded a triarch-tetrarch actynostele in developing protocorms, but it should be remembered that the organization of the vascular system undergoes changes during plant development [81].

Additionally, leaf characteristics are in line with the ecological adaptations of *C. purpurata* to a tropical environment. The roughness and thickness of *C. purpurata* leaf cuticles are consistent with characters and data previously annotated for Laeliinae [67]. The cuticle, relatively thick if compared to other orchid species [82], plays an ecological role in transpiration reduction and in drought resistance [83–85]. Similar considerations could be drawn for stomata dimensions, as small stomata are known to be quicker to close and then are more tolerant to dry conditions [84].

The stomatal apparatus is tetracytic, as noted in most Laeliinae; however, while a reniform shape was described for all the other taxa [66,67], the C-shaped guard cells that we observed were previously reported only for *Cattleya sincorana* (syn. *Sophronitis sincorana*). This latter record can be useful to corroborate taxonomical information regarding the studied species.

The data regarding the discontinuous hypodermis, the homogeneous mesophyll, and the dorsiventral one in correspondence of the central rib, together with the characteristics of vascular system, agree to what is generally known for Laeliinae [67]. On the other hand, the presence of transversally developed elements, which constitute anastomoses between veins in the mesophyll, was less frequently annotated in the subtribe. It has been proposed that they provide mechanical stability and discourage herbivory [86]. The occurrence of transverse veins in the mesophyll of *C. purpurata* is in line with previous findings for the genus *Cattleya* [87].

#### **5. Conclusions**

In conclusion, we compared germination on different media and, for the first time, attempted symbiotic germination for *Cattleya purpurata*, demonstrating the potential use of Tulasnellaceae, which otherwise need to be refined, looking for the best symbiotic fungal partner because germination percentage was lower than that obtained on some asymbiotic media. Furthermore, we characterized its seed and seedling morphology, providing new insights into the knowledge of this endangered species. Seedling morphology was comparable with other congeneric species, showing typical characteristics of epiphytic orchids such as root velamen with 2–3 cells layers. We confirmed the presence of tilosomes in roots in this orchid genus and observed stomata with C-shaped guard cells in leaves, which are unusually found in *Cattleya* species. Interestingly, we pointed out the presence of calcium oxalate crystals, both in idioblasts as raphides (in roots and leaves) and in the seed suspensor as druzes. It would be intriguing, in the future, to see if other orchid species actually accumulate these crystals in the suspensor and if they have a nutritional/functional role in the orchid symbiosis.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/horticulturae7110480/s1, Figure S1: Correlation between the volume of the embryo and the seed analyzed by means of the cor.test( ) function using the Spearman's rank correlation coefficient rho, Figure S2: Main elements present in crystals determined by SEM-EDS spectrum. The analysis showed a chemical composition typically obtained for calcium oxalate, with carbon (C), oxygen (O) and calcium (Ca) peaks.

**Author Contributions:** Conceptualization, J.C. and M.B.; methodology, J.C., M.B., A.C. and L.C.; software, J.C., M.B. and M.A.; validation, J.C., M.B. and L.C.; formal analysis, J.C. and M.B.; investigation, J.C. and M.B.; resources, J.C., L.C. and A.G.; data curation, J.C. and M.B.; writing—original draft preparation, J.C. and M.B.; writing—review and editing, M.B., J.C., M.A., A.G., A.C. and L.C.; visualization, J.C., M.B., M.A., A.C. and L.C.; supervision, L.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The experimental work for this study has been performed within the framework of the doctoral research projects of J. Calevo (PhD School in Biology and Applied Biotechnologies, University of Turin) and M. Bazzicalupo (PhD School in Science and Technology for the Environment and Territory, University of Genoa), both funded by the Ministry of Education and Research (MIUR). M. Adamo was supported by a grant from CMIRA (Coopération et Mobilité Internationales Rhône-Alpes) program of region Rhône-Alpes, France. We thank Laura Negretti for SEM images.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Author details**

C2V Company released its first commercial micro-GC product (model C2V-200) in 2010. The platform, based on cartridges, allows hybrid integration of components such as sensors and valves to be assembled together with reduced dead volume. Integrated microchip technology combined with narrow bore capillary GC columns results in a higher performance for lower costs. The C2V-200 micro-GC is designed for ease of use, reduced maintenance, and low gas consumption. Exchangeable column cartridges, with integrated heating zones, can be easily installed (up to 4) with a different column and detection method. This modular setup allows the ability to monitor a wider spectrum of gas components in the same timeframe of 10 to

The concept of micro-gas chromatographs demonstrates the potential of mobile devices in various fields related to analytical chemistry such as oil and gas, air analyses, defense, food processing industry, etc. New instrument designs and component manufacturing methods are coming on line that will result in the development of a new generation of high-performance, moveable, and miniaturized instruments for high-performance gas chromatography (HP-GC). The use of microelectromechanical system technologies for the manufacturing of microfabricated gas chromatographic components results in very small, autonomous, and low-cost instruments. Completely autonomous GC instruments require no daily maintenance and can be placed in remote locations for long-term service. This requires battery operation, wireless communications, and freedom from tanks of compressed gases. To this end, work is in progress to develop a high-performance micro-GC that will have acceptable volume. To achieve complete autonomy, vacuum outlet GC should be used with ambient air as carrier gas. In addition, remote battery charging with radiofrequency transmission will be feasible. The use of ambient air as a carrier gas poses several challenges. First, some stationary phases rapidly decompose in air. Poly(ethylene glycol) is a good example. In addition, particulate material and water vapor may need to be removed. Sensor array detection also is needed because these devices can be microfabricated with very low dead volumes; they require no support gases for their operation, and they can be fabricated with a variety of selectivities, which can be used for vapor recognition and for the deconvolution of overlapping peaks. This can reduce the resolution requirements for the column. Sensors and detectors usually have lower sensitivity than detectors incorporated in laboratory gas chromatographic instruments. Low detector sensitivity, coupled with the very low concentrations often associated with air monitoring, requires the use of a preconcentrator for sample enrichment prior to separation and detection. More energy is then needed to heat the preconcentrator to release the adsorbed sample. It seems that one way to solve these problems is a future integration of the instrument on a single chip, focusing each device to one field of application instead to make universal

apparatus emulating conventional gas chromatographs.

The authors declare no conflict of interest.

**Conflict of interest**

60 seconds [50].

164 MEMS Sensors - Design and Application

**7. Conclusion**

Imadeddine Azzouz1,2\* and Khaldoun Bachari1