1. Introduction {#sec1}
===============

The question what distinguishes human from other animals and especially from other primates \[[@r1]\] is one of the driving forces of the scientific interest in evolution in general. There are many ways to approach this question, like comparison of anatomy, etiology, behavior, or genetics, to mention only a few possibilities \[[@r2]\]. The genetics of different species can be compared on different levels of resolution, like classical and banding cytogenetics, molecular cytogenetics or molecular genetics. While (molecular) cytogenetics leads to resolution levels of 2-10 megabasepairs, molecular genetics can go down to the DNA- and basepair level. However, molecular genetics, esp. sequencing approaches, cannot analyze repetitive regions of genomes, constituting bog parts of genomes, also being considered as potentially important for speciation. Thus (molecular) cytogenetic and molecular genetic data complement each other and both are needed for deep understanding of evolutionary changes \[[@r3], [@r4]\].

1.1. Cytogenetics and Molecular Cytogenetics {#sec1.1}
--------------------------------------------

Classical and banding cytogenetic data is available for most Old World Monkeys (OWMs), while detailed molecular (cyto)genetic data is in general sparse. Here, seven OWM-species were studied by means of fluorescence *in situ* hybridization (FISH)-banding \[[@r5]\] and locus-specific probes and compared to each other and with data from the literature. Those were from Africa *Macaca sylvanus* (MSY) and *Mandrillus sphinx* (MSP) and from South East Asia *Macaca arctoides* (MAR) *M. fascicularis* (MFA), *M. nemestrina* (MNE), *M. assamensis* (MAS) and *M. mulatta* (MMU).

For macaques (Catarrhini; Ceropithecoidae) being a morphologically highly diverse group, a quick radiation during the last 3-5 million years in Africa and especially Asia is suggested \[[@r6]\]. Cytogenetic data was available for them as summarized in Table **[1](#T1){ref-type="table"}**, indicating for 20 autosome pairs and two heteromorphic gonosomes in males of these species \[[@r7]\]. Also, most important (FISH) and molecular genetic studies previously available for the seven studied species are summarized in Table **[1](#T1){ref-type="table"}**.

Here the first comparative molecular cytogenetic study for the characterization of the karyotype of six macaque species and mandrill using human multicolor banding combined with locus-specific and heterochromatin-specific probes is presented.

2. Material and Methods {#sec2}
=======================

2.1. Cell Culture and Chromosomal Preparation {#sec2.1}
---------------------------------------------

Immortalized male and female lymphoblast cell lines derived from stump-tailed macaque (*Macaca arctoides*, MAR), crab-eating macaque (*Macaca fascicularis*, MFA), southern pig-tailed macaque (*Macaca nemestrina*, MNE), Assam macaque (*Macaca assamensis*, MAS), barbary macaque (*Macaca sylvanus*, MSY), rhesus macaque (*Macaca mulatta*, MMU) and mandrill (*Mandrillus sphinx*, MSP) were cultivated according to standard techniques. Chromosomes were prepared following standard protocols \[[@r36]\].

2.2. Fluorescence *In Situ* Hybridization (FISH) {#sec2.2}
------------------------------------------------

FISH was done as previously reported using human derived MCB probe sets or locus-specific bacterial artificial chromosomes (BAC) probes, in parts combined as subcentromere/subtelomere-specific multicolor (subCTM-)FISH probe sets \[[@r37], [@r38]\]. Additionally, three *Homo sapiens* (HSA) derived homemade microdissection probes were utilized: a probe specific for the short arm of all human acrocentric chromosomes, and others for 1q12 and 9q12, 9p12/ 9q13, 16q11.2 and Yq12 \[[@r39]\].

Images were captured by an Axioplan II microscope (Carl Zeiss Jena GmbH, Germany) equipped with filter sets for DAPI, FITC, TR, SO, Cy5 and DEAC. Image analysis was performed *via* pseudocolor banding and fluorochrome profile analyses using the ISIS digital FISH imaging system (MetaSystems Hard & Software GmbH, Altlussheim, Germany). A total of 10 up to 20 metaphases per species and probe were taken into account.

3. Results {#sec3}
==========

The karyotypes of all here studied seven species were on molecular cytogenetic identical. The detected changes compared to human karyotype are summarized in Table **[2](#T2){ref-type="table"}** \[[@r40]\]. Also [Fig. (**[1](#F1){ref-type="fig"}**)](#F1){ref-type="fig"} summarizes the results obtained for all species exemplified for MAR.

Overall, 11 inversions, 10 neocentromere formations and two translocation events were observed with respect to the human karyotype. Besides, chromosomes being homologous to human chromosomes 3, 6, 9, 17 and 21 had highly complex rearrangements not simply to explain or describe by inversions or insertions. Furthermore, repetitive DNA was identified as follows:

-   repetitive sequence D1Z5 located in HSA in 1q11-q12 was present in all studied species at the corresponding homologous region on their chromosome 1;

-   the human hemiheterochromatic region 9p12/ 9q13 is located on long arm of monkey chromosomes 15, while D9Z3 in from HSA 9q12 is not detectable in the studied species;

-   the region being present in human as band 16q11.2 (D16Z3) could also be found in the studied OWMs at the homologous region on monkey chromosome 20;

-   the region being present in human 10 times at the short arms of the acrocentric chromosomes can only be found at the long arm of chromosome 10 distal to the Nucleolus Organizer Region (NOR) and the centromere of this chromosome; and

-   unknown, monkey specific DNA was amplified and located in regions homologous to HSA 9q33.2, 17p10, 17q24, 18q21.1 and Yqter, distal to the telomeric sequences. Repetitive DNA as present in human male in Yq12 was not observed in the studied OWMs.

According to Table **[3](#T3){ref-type="table"}**, 33 of 51 evolutionary conserved breakpoints appearing in the seven studied species, *i.e.* 65% colocalize with fragile sites.

4. Discussion {#sec4}
=============

The present study is another good example for suitability of molecular cytogenetics, especially MCB combined with locus-specific and heterochromatin-specific probes, to get new insights into chromosomal evolution of primates. Previous comparable studies were done in *Gorilla gorilla* \[[@r36]\], *Hylobates lar* \[[@r43]\] and *Trachypithecus cristatus* \[[@r38]\]. In those studies more or less unique karyotypic features were observed, while in the present one surprisingly the identical karyotype was found in seven species of OWM. Overall, this result is in concordance with previous cytogenetic studies at lower resolution (Table **[1](#T1){ref-type="table"}**). The here described evolutionary conserved inv(4)(p15.3q10) was initially only reported by Karere *et al.* \[[@r44]\], however with other suggested breakpoints. Furthermore, the karyotypic uniformity of the studied species is confirmed also by the fact that for some of them interspecies crossing was reported in captivity \[[@r45], [@r46]\] and also in common ancestors as recent sequence analysis between macaque species groups imply \[[@r47]\].

Compared *e.g.* to *Hylobates lar* \[[@r43]\], there are only few evolutionary conserved breakpoints and rearrangements present in macaques and mandrill compared to HSA. Still the 'complex rearranged' chromosomes homologous to human chromosomes 3, 6, 9, 17 and 21, being afterwards stable during evolution is striking and completely different than observed *e.g.* in New World Moneys (own unpublished data).

Repetitive elements may also play their role in speciation -- here macaque and mandrill specific DNA-amplifications could be found for previously described regions like that being homologous to human 18q21.1 \[[@r48]\]. Also observations on seeding of neocentromeric regions preferentially in gene deserts \[[@r49]\] fit to the here described data (Table **[2](#T2){ref-type="table"}**). As others suggested before \[[@r50]\] we could also confirm a high degree of colocalization of fragile sites with the here reported evolutionary conserved breakpoints.

In this special group of OWMs karyotypic evolution cannot be the driving force of speciation. Thus one can expect submicroscopic genetic changes in the genomes of the seven here studied and phenotypically so different species as described *i.e.* by Yan *et al.* \[[@r51]\] for MMU subspecies and MFA. Among the evolutionary forces leading to annidation of the studied species, spatial separation, may be an explanation for a part of the speciations, like for African *M. sylvanus*, *Mandrillus sphinx* on the one and the South Asian macaques on the other end. However, for the five studied South Asian macaques partially or completely overlapping in habitats the species separation process might must have other reasons. One idea for a driving force comes from recent paper of Zhou *et al.* \[[@r52]\] suggesting niche separation of *M. assamensis* and *M. mulatta* based on adaptation to reduce resource competition.

CONCLUSION
==========

Even though karyotypic evolution plays a major role in speciation and species separation this seems to be unimportant between the seven here studied Catarrhini-species. Submicroscopic changes, like gene mutations, activation of pseudogenes, *etc*. seem to be the main reasons for the phenotypic differences of those species.

Supported in parts by the China Scholarship Council (support for FX), a "Thai Government Science and Technology Scholarship" for KP, a "Strategic scholarship fellowships frontier research network for SP and the DLR/BMBF RUS 09/008 (AW). WS received financial support from the Thailand Research Fund through the Royal Golden Jubilee Ph.D. program joint funding with Khon Kaen University (Grant No. PHD/0081/2556).

AIDS

:   Acquired Immune Deficiency Syndrome

BAC

:   Bacterial Artificial Chromosomes

cen

:   Centromeric position

COPOG

:   Cloning of Parts of Genome

DNA

:   Deoxyribonucleic acid

FB

:   FISH-banding

FISH

:   Fluorescence *in situ* hybridization

HSA

:   *Homo sapiens*

LSP

:   Locus Specific Probes

MAR

:   *Macaca arctoides*

MAS

:   *Macaca assamensis*

MCB

:   Multicolor-banding

MFA

:   *Macaca fascicularis*

MMU

:   *Macaca mulatta*

MNE

:   *Macaca nemestrina*

MSP

:   *Mandrillus sphinx*

MSY

:   *Macaca sylvanus*

NGS

:   Next Generation Sequencing

NOR

:   Nucleolus Organizer Region

OWM

:   Old World Monkey

SAS

:   Sanger sequencing

subCTM-FISH

:   Subcentromere/subtelomere-specific multicolor fluorescence *in situ* hybridization

WCP

:   Whole Chromosomes Paints

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
==========================================

Not applicable.

HUMAN AND ANIMAL RIGHTS
=======================

No Animals/Humans were used for studies that are base of this research.

CONSENT FOR PUBLICATION
=======================

Not applicable.

CONFLICT OF INTEREST
====================

The authors declare no conflict of interest, financial or otherwise.

![Results of MCB and selected locus- and heterochromatin-specific probes are depicted here. Macaque chromosomes are numbered according to Morescalchi *et al.* \[[@r29]\].](CG-19-207_F1){#F1}

###### 

Previous studies done in the here studied OWM-species.

  ----------------------------------------------------------------------------------------------------------------------------------
  **Methods Species**          **Cytogenetics**           **Molecular Cytogenetics**   **Molecular Genetics**
  ---------------------------- -------------------------- ---------------------------- ---------------------------------------------
  ***Macaca arctoides***\      \[[@r7]-[@r10]\]           n.a.                         SAS \[[@r11]\]
  **MAR**                                                                              

  ***Macaca fascicularis***\   \[[@r7], [@r12]-[@r15]\]   LSP \[[@r16]-[@r20]\]\       COPOG \[[@r23]\]\
  **MFA**                                                 WCP \[[@r21]\]\              SAS \[[@r11], [@r24]\]
                                                          FB \[[@r22]\]                

  ***Macaca nemestrina***\     \[[@r7]\]                  FB \[[@r25]-[@r26]\]         SAS \[[@r11]\]
  **MNE**                                                                              

  ***Macaca assamensis***\     n.a.                       n.a.                         SAS \[[@r27]-[@r28]\]
  **MAS**                                                                              

  ***Macaca sylvanus***\       \[[@r29]\]                 WCP \[[@r29]\]\              SAS \[[@r11]\]
  **MSY**                                                 FB \[[@r30]\]                

  ***Macaca mulatta***\        \[[@r7], [@r13]\]          LSP \[[@r31]-[@r33]\]\       SAS \[[@r3], [@r11], [@r33]\] NGS \[[@r3]\]
  **MMU**                                                 WCP \[[@r34]\]               

  ***Mandrillus sphinx***\     \[[@r15]\]                 LSP \[[@r19]\]               SAS \[[@r35]\]
  **MSP**                                                                              
  ----------------------------------------------------------------------------------------------------------------------------------

Abbreviations: COPOG = Cloning of Parts of Genome; FB = FISH-banding; LSP = Locus Specific Probes; n.a. = Not Available; NGS = Next Generation Sequencing; SAS = Sanger Sequencing; WCP = Whole Chromosomes Paints.

###### 

Breakpoints of macaques according to MCB and molecular data from Ventura *et al.* \[[@r40]-[@r41]\].

  ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  **HSA-MCB-probe**                              **MAR / MNE / MAS / MFA / MMU / MSP / MSY**                                                                               **Breakpoint position \[NCBI36/ hg18\]**   **BACs**
  ---------------------------------------------- ------------------------------------------------------------------------------------------------------------------------- ------------------------------------------ ----------------------------
  **1**                                          inv(1)(q23.3q42.13)                                                                                                       160,918,751-\                              RP11-572K18 + RP11-331H2
                                                                                                                                                                           161,225,664                                

  cen in 1q42.13                                 226,810,735-226,866,653                                                                                                   RP4-621015                                 

  **2**                                          **inv(2)(q11.1q14.1)**\                                                                                                   89,772,752 -95,469,732                     RP11-468G5/ RP11-316G9
                                                 in bold acc. to online resource Uni Bari                                                                                                                             

  114,076,736-114,076,791                        n.a.                                                                                                                                                                 

  cen in 2p11.2 in M-13                          86,622,638-86,827,260                                                                                                     RP11-722G17                                

  **inv(2)(q14.1q21.1)**\                        114,076,736-114,076,791                                                                                                   n.a.                                       
  in bold acc. to online resource Uni Bari                                                                                                                                                                            

  131,799,777-131,995,056                        RP11-109E12                                                                                                                                                          

  cen in 2q22.1 in M-12                          138,730,526-138,830,121                                                                                                   RP11-846E22\                               
                                                                                                                                                                           RP11-343I5                                 

  **3**                                          der(3)(qter-\>q27.3::**p22.3-\>p24**::q22.1-\>q27.3::p22.3-\>p12.3::p26.3-\>p24::q22.1-\>p12.3:)\                         0-\                                        RP11-183N22
                                                 in bold acc. to online resource Uni Bari                                                                                  4,328,222                                  

  15,045,785-15,213,797                          RP11-616M11                                                                                                                                                          

  36,506,239-36,658,135                          RP11-240N7                                                                                                                                                           

  75,628,601-75,698,634                          RP11-634L22/ RP11-413E6                                                                                                                                              

  131,347,36-131,354,303                         RP11-787P10/ RP11-924M2                                                                                                                                              

  187,819,875-187,998,697                        RP11-177B11                                                                                                                                                          

  cen in 3q26.1                                  164,122,697-164,539,723                                                                                                   RP11-355I21/ RP11-418B12                   

  **4**                                          inv(4)(p15.3q10)                                                                                                          86,039,028-86,261,868                      RP11-367P3

  48,773,495-52,354,875                          RP11-317G22/ RP11-365H22                                                                                                                                             

  cen identical                                  \-                                                                                                                        \-                                         

  **5**                                          no change                                                                                                                 \-                                         \-

  cen: identical                                 \-                                                                                                                        \-                                         

  **6**                                          inv(6)(p24q25.2) and inv(6)(q21q25.2)                                                                                     0-\                                        subtelomeric probe (Vysis)
                                                                                                                                                                           213,636                                    

  108,439,777-108,647,294                        RP11-815N24                                                                                                                                                          

  158,977,778-159,193,482                        RP11-507C10                                                                                                                                                          

  cen in 6q24.3                                  145,651,644-145,845,896                                                                                                   RP11-474A9                                 

  **HSA-MCB-probe**                              **MAR / MNE / MAS / MFA / MMU / MSP / MSY**                                                                               **Breakpoint position \[NCBI36/ hg18\]**   **BACs**

  **7**                                          der(7)(21qter-\>21q11.2::**7p22.3-\>7p22.1::7q21.3-\>7q22.1**::7q11.23-\>7p**21.3::7p21.3-\>7q11.23::7q22.1-**\>7qter)\   6,613,748-7,043,428                        RP11-108O03/ RP11-1061P7
                                                 in bold acc. To online resource Uni Bari                                                                                                                             

  76,490,507-76,700,668                          RP11-606M6                                                                                                                                                           

  97,263,693-97,536,166                          RP11-652L7/ RP11-150J17                                                                                                                                              

  101,859,446-103,221,699                        RP11-163E9/ RP11-418B19                                                                                                                                              

                                                 cen identical                                                                                                             \-                                         \-

  **8**                                          no change                                                                                                                 \-                                         \-

  cen identical                                  \-                                                                                                                        \-                                         

  **9**                                          der(9)(9qter-\>9q34::?-\>?::9q34-\>9p24.3::9q21.11-\>9q22.33☺                                                             0-\                                        RP11-3J10
                                                                                                                                                                           615,148                                    

  \-                                             \-                                                                                                                                                                   

  70,000,000-70,488,561                          HAS band 9q12/\                                                                                                                                                      
                                                 RP11-203L2                                                                                                                                                           

  98,602,467-98,954,600                          RP11-330M2 + RP11-520B13                                                                                                                                             

  del(9)(q12q12)                                 \-                                                                                                                        \-                                         

  cen in 9q33.2                                  124,189,785-124,493,134                                                                                                   RP11-542K23/ RP11-64P14                    

  unknown material in 9q33.2                     \-                                                                                                                        \-                                         

  **10**                                         inv(10)(q11.23q22.3)                                                                                                      52,020262-52,248,654                       RP11-591H22

  88,943,287-89,105,572                          RP11-322M19                                                                                                                                                          

  cen identical                                  \-                                                                                                                        \-                                         

  **11**                                         inv(11)(p15.4q13.4)                                                                                                       3,455,204-\                                RP11-650F7/\
                                                                                                                                                                           3,501,436                                  RP11-749O23

  71,060,796-71,133,202                          RP11-684B2/ RP11-483L13                                                                                                                                              

  cen in 11p15.4                                 5,864,725-5,865,181                                                                                                       RP11-625D10/ RP11-6661M13                  

  **12**                                         no change                                                                                                                 \-                                         \-

  cen: identical                                 \-                                                                                                                        \-                                         

  **13**                                         no change                                                                                                                 \-                                         \-

  cen in 13q21.31                                61,282,357-61,709,544                                                                                                     RP11-1043D14 + RP11-539I23                 

  **14**                                         der(15)t(14;15)(q11.2;q26.3)                                                                                              prox. From 18,806,381                      RP11-324B11

  cen see [@r15]                                 \-                                                                                                                        \-                                         

  **HSA-MCB-probe**                              **MAR / MNE / MAS / MFA / MMU / MSP / MSY**                                                                               **Breakpoint position \[NCBI36/ hg18\]**   **BACs**

  **15**                                         der(15)t(14;15)(q11.2;q26.3)                                                                                              18,400,000-22,905,050                      centromere\
                                                                                                                                                                                                                      RP11-441B20

  cen in 15q25                                   82,835,478-83,006,963                                                                                                     RP11-182J1                                 

  **16**                                         **inv(16)(q22.1q22.3)**\                                                                                                  68,394,830-68,894,008                      RP11-779G13/ RP11-155G24
                                                 in bold acc. to online resource Uni Bari                                                                                                                             

  72,719,303-73,147,016                          RP11-339I16/ RP11-236J9                                                                                                                                              

  dim(16)(q11.2)                                 \-                                                                                                                        \-                                         

  cen identical                                  \-                                                                                                                        \-                                         

  **17**                                         der(17)(pter-\>q12::**q23.3-\>q21.32::q12-\>q21.32**::q23.3-\>qter)\                                                      33,322,352-33,713,298                      RP11-115K3/ RP11-923C2
                                                 in bold acc. to online resource Uni Bari                                                                                                                             

  42,866,560-43,587,728                          RP11-671B19/ RP1142F20                                                                                                                                               

  57,597,398-57,765,687                          RP11-42F20/ RP11-50G1                                                                                                                                                

  unknown material in 17p10 and 17q24 inserted   \-                                                                                                                        \-                                         

  cen identical                                  \-                                                                                                                        \-                                         

  **18**                                         no change                                                                                                                 \-                                         \-

  cen in 18q21.2                                 50,313,129-50,360,135                                                                                                     RP11-61D1/ RP11-289E15                     

  unknown material in 18q21.1                    \-                                                                                                                        \-                                         

  **19**                                         no change                                                                                                                 \-                                         \-

  cen identical                                  \-                                                                                                                        \-                                         

  **20**                                         der(20)(22qter-\>22p13::20p11.21-\>20p13::20q11.21-\>20qter)                                                              25,522,225-29,667,570                      RP11-694B14/ RP5-854E16

  0-\                                            RP11-530N10                                                                                                                                                          
  659,205                                                                                                                                                                                                             

  cen see [@r22]                                 \-                                                                                                                        \-                                         

  **21**                                         der(7)(21qter-\>21q11.2::**7p22.3-\>7p22.1::7q21.3-\>7q22.1**::7q11.23-\>**7p21.3::7p21.3-\>7q11.23::7q22.1-**\>7qter)\   13,200,000 14,822,550                      centromere\
                                                 in bold acc. to online resource Uni Bari                                                                                                                             RP11-641G16

  cen see [@r7]                                  \-                                                                                                                        \-                                         

  **22**                                         der(20)(22qter-\>22p13::20p11.21-\>20p13::20q11.21-\>20qter)                                                              14,430,000-16,159,326                      centromere\
                                                                                                                                                                                                                      CTA-115F6

  cen identical with HSA22                       \-                                                                                                                        \-                                         

  **X**                                          no change                                                                                                                 \-                                         \-

  cen identical                                  \-                                                                                                                        \-                                         

  **Y**                                          del(Y)(q12q12)                                                                                                            \-                                         \-

  cen identical                                  \-                                                                                                                        \-                                         

  unknown material in Yqter                      \-                                                                                                                        \-                                         
  ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Abbreviations: cen = centromeric position.

###### 

Evolutionary conserved breakpoints in the seven studied species compared with human fragile sites (FS). Data on FS localizations are listed acc. to Mrasek *et al.* \[[@r42]\].

  **Breakpoint in Studied OWMs**   **Fragile Site**   **Breakpoint in Studied OWMs**   **Fragile Site**
  -------------------------------- ------------------ -------------------------------- ------------------
  1q23.3                           FRA1P              7q22.1                           FRA7F
  1q42.13                          FRA1H              9p24.3                           FRA9H
  2p11.2                           FRA2L              9q21.11                          FRA9D
  2q11.1                           FRA2R              9q22.33                          n.a.
  2q14.1                           n.a.               9q33.2                           FRA9M
  2q21.1                           n.a.               9q34                             FRA9N
  2q22.1                           n.a.               10q11.23                         FRA10J
  3p26.3                           FRA3E              10q22.3                          n.a.
  3p24                             FRA3A              11p15.4                          FRA11J
  3p22.3                           FRA3G              11q13.4                          FRA11E
  3p12.3                           FRA3I              13q21.31                         n.a.
  3q22.1                           FRA3N              14q11.2                          FRA14D
  3q26.1                           FRA3O              15q25                            FRA15F
  3q27.3                           FRA3C              15q26.3                          FRA15G
  4p15.3                           FRA4D              16q22.1                          FRA16C
  4q10                             n.a.               16q22.3                          n.a.
  6p24                             n.a.               17q12                            FRA17D
  6q25.2                           n.a.               17q21.32                         n.a.
  6q21                             FRA6F              17q23.3                          n.a.
  6q24.3                           n.a.               18q21.2                          FRA18B
  6q25.2                           FRA6M              20p13                            FRA20C
  7p22.3                           FRA7B              20p11.21                         n.a.
  7p22.1                           n.a.               20q11.21                         n.a.
  7p21.3                           FRA7L              21q11.2                          FRA21
  7q11.23                          FRA7J              22p13                            n.a.
  7q21.3                           n.a.               \--                              \--
