Skip to main content
NCBI home page
Zoological Studies logoLink to Zoological Studies
. 2020 Mar 3;59:e5. doi: 10.6620/ZS.2020.59-5

The Highest Chromosome Number and First Chromosome Fluorescent in situ Hybridization in the velvet worms of the family Peripatidae

Dutra Débora Duarte 1, Lucas Henrique Bonfim Souza 1, Lívia Medeiros Cordeiro 1, Douglas Araujo 1,*
PMCID: PMC7184263  PMID: 32346453

Abstract

The diversity of Onychophora is poorly studied, despite there being nearly 200 described species divided in two families: Peripatidae and Peripatopsidae. Peripatid velvet worms are found mainly in the Neotropical region. The low morphological diversity in Peripatidae is an obstacle to determining its taxonomy, and chromosomal analyses can help clarify this. The aim of this work was to chromosomally analyze one species of Epiperipatus from Mato Grosso do Sul, Brazil. Conventional staining and telomeric fluorescent in situ hybridization (FISH) were performed with the gonads of three males of Epiperipatus sp. The specimens showed 2n♂ = 73, the largest diploid number found in Onychophora to date, with the majority of chromosomes acro/telocentrics and the largest element submetacentric. The FISH marked the telomeric region of all elements and revealed one Interstitial Telomeric Site (ITS) on the proximal region of the long arm large submetacentric chromosome. The absence of male meiosis and female cell division in the analyzed specimens prevented us from determining whether the unpaired large submetacentric is a sex chromosome, which could lead to the description of a rare sex chromosome system (SCS) in Onychophora, or a case of fusion between autosomes. In either case, the presence of ITS is a clear indication of chromosomal fusion.

Keywords: Cryptic species, Epiperipatus, Karyotype, Heteromorphic chromosome, Interstitial Telomeric Site

BACKGROUND

Onychophora are ancient Panarthropod animals with soft bodies, lobopodial legs with claws and a peculiar hunting strategy by which they eject glue to capture prey (Monge-Najera 1995). The group has 201 species distributed within Peripatidae (81 species) and Peripatopsidae (120 species), 20 of which are considered nomina dubia; however, the biodiversity of these species is far from established (Oliveira et al. 2020).

Most peripatids are from Neotropical environments, and the Brazilian velvet worm fauna consists of only 16 species, four of which are considered vulnerable or endangered (Oliveira et al. 2015; Instituto Chico Mendes de Conservação da Biodiversidade 2018). Furthermore, peripatids include several other Brazilian species that are undescribed (Sampaio-Costa et al. 2009; Oliveira et al. 2010). One of the main reasons they are undescribed is that their characters have low diversity, making it difficult to efficiently compare species (Sampaio-Costa et al. 2009; Oliveira et al. 2012a).

Besides the taxonomic gaps mainly in Peripatidae, several studies have found cryptic species of Onychophora, including in Brazilian species of Epiperipatus (Clark, 1913) (Reid et al. 1995; Reid 1996; Lacorte et al. 2011; Oliveira et al. 2011 2018). Cryptic speciation and narrow geographical range are expected due to their low dispersal, cryptic habitat and reproductive biology (New 1995; Oliveira et al. 2011 2014).

Both families have few cytogenetic analyses, with Peripatidae being less studied, with only 6.17% of the species already karyotyped (Table 1) (Oliveira et al. 2012b). The lowest and highest diploid numbers found were 2n♂ = 8 in Eoperipatus sp. and 2n♂ = ± 60 in Epiperipatus biolleyi (Bouvier, 1902), both belonging to Peripatidae (Mora et al. 1996; Jeffery et al. 2012; Oliveira et al. 2012b). Among species with a described Sex Chromosome System (SCS), the most common is the type ♂XY (Table 1), except for a population of Euperipatoides rowelli (Reid, 1996) that presented the type ♂X1X2Y (Rowell et al. 1995). Studies with differential chromosome techniques are virtually absent for velvet worms. The C-banding technique does not work for the group, and only one study employing chromosome fluorescent in situ hybridization (FISH) technique has been performed so far, showing (TTAGGG)n telomeric repeats only on terminal ends of pachytene bivalents of Peripatopsis stelliporata Sherbon & Walker, 2004 (Peripatopsidae), despite their importance for understanding the chromosome evolution in the clade (Rowell et al. 1995 2002; Vítková et al. 2005; Jeffery et al. 2012; Oliveira et al. 2012b 2018).

Table 1.

Review of Onychophora cytogenetics. Diploid number (2n♂), Sex Chromosome System (SCS), chromosomal morphology, locality and references

Peripatidae
Species 2n♂ SCS Chromosomal morphology Locality Reference
Cerradopatus sucuriuensisa Oliveira et al., 2015 (cited as Epiperipatus sp.) 22 - - Brazil Jeffery et al. 2012
C. sucuriuensis Oliveira et al., 2015 22 - 6 acrocentric, 5 metacentric /submetacentric Brazil Oliveira et al. 2015
Eoperipatus sp. 8 - - Thailand Jeffery et al. 2012
Eoperipatus sp. 8 - - Thailand Oliveira et al. 2012b
Epiperipatus biolleyi (Bouvier, 1902) ± 60b - - Costa Rica Mora et al. 1996
Epiperipatus sp. 73 - 1 submetacentric, 72 acro/telocentric Brazil Present study
Principapillatus hitoyensis Oliveira et al., 2013 54 XY - Costa Rica Jeffery et al. 2012
P. hitoyensis Oliveira et al., 2013 54 XY 17 acrocentric, 9 metacentric/submetacentric and XY acrocentric Costa Rica Oliveira et al. 2012b
Centorumis trigonac Reid, 1996 (cited as Euperipatoides sp.) 26 - - Australia Rowell et al. 1995
Cephalofovea cameroni Reid et al., 1995 28 - - Australia Reid et al. 1995
Cephalofovea clandestina Reid et al., 1995 28 - - Australia Reid et al. 1995
Cephalofovea pavimenta Reid et al., 1995 34 - - Australia Reid et al. 1995
Cephalofovea tomahmontis Ruhberg, 1988 34 XY - Australia Reid et al. 1995
C. tomahmontis Ruhberg, 1988 34 XY - Australia Rowell et al. 1995
Diemenipatus mesibovi Oliveira et al., 2018 18 XY - Tasmania Oliveira et al. 2018
Diemenipatus taiti Oliveira et al., 2018 18 - - Tasmania Oliveira et al. 2018
Euperipatoides kanangrensisc Reid, 1996 (cited as Euperipatoides sp.) 32 XY - Australia Rowell et al. 1995
E. kanangrensis Reid, 1996 32 XY - Australia Jeffery et al. 2012
Euperipatoides leuckartii (Sänger, 1871) 32 XY - Australia Rowell et al. 1995
Euperipatoides rowelli Reid, 1996 34 XY - Australia Jeffery et al. 2012
Euperipatoides rowellic Reid, 1996 (cited as Euperipatoides sp.) 33 X1X2Y - Australia Rowell et al. 1995
E. rowellic Reid, 1996 (cited as Euperipatoides sp.) 34 - - Australia Rowell et al. 1995
E. rowellic Reid, 1996 (cited as Euperipatoides sp.) 34 XY - Australia Rowell et al. 1995
Euperipatoides sp. 18 XY - Tasmania Rowell et al. 1995
Euperipatoides sp. 18 - - Tasmania Rowell et al. 1995
Leucopatus anophthalmus Oliveira et al., 2018 36 - - Tasmania Oliveira et al. 2018
Nodocapitus inornatusc Reid, 1996 (cited as Euperipatoides sp.) 30 - - Australia Rowell et al. 1995
Ooperipatellus insignis (Dendy, 1890) 42 XY Acrocentric, metacentric and telocentric Australia Rowell et al. 2002
Ooperipatellus nickmayeri Oliveira and Mayer, 2017 50 XY - Tasmania Oliveira and Mayer 2017
Ooperipatellus sp. 1 42 - Acrocentric, metacentric and telocentric Australia Rowell et al. 2002
Ooperipatellus sp. 2 42 - Acrocentric, metacentric and telocentric Tasmania Rowell et al. 2002
Ooperipatus hispidus Reid, 1996 22 XY - Australia Jeffery et al. 2012
Peripatopsis balfouri (Sedgwick, 1885) (cited as Peripatus balfouri) 28 - - South Africa Montgomery 1900
Phallocephale tallagandensis Reid, 1996 18 XY - Australia Jeffery et al. 2012
Planipapillus biacinaces Reid, 1996 40 - Telocentric Australia Rowell et al. 2002
Planipapillus bulgensis Reid, 1996 24 - Metacentric, submetacentric and telocentric Australia Rowell et al. 2002
Planipapillus cyclus Reid, 2000 26 Metacentric, submetacentric and telocentric Australia Rowell et al. 2002
Planipapillus impacris Reid, 2000 30 - Metacentric, submetacentric and telocentric Australia Rowell et al. 2002
Planipapillus mundus Reid, 1996 40 - Telocentric Australia Rowell et al. 2002
Planipapillus taylori Reid, 1996 38 - Metacentric, submetacentric and telocentric Australia Rowell et al. 2002
Planipapillus sp. 1 22 - Metacentric, submetacentric and telocentric Australia Rowell et al. 2002
Planipapillus sp. 2 20 - Metacentric Australia Rowell et al. 2002
Planipapillus sp. 3 32 - Metacentric, submetacentric and telocentric Australia Rowell et al. 2002
Planipapillus sp. 4 32 - Metacentric, submetacentric and telocentric Australia Rowell et al. 2002
Planipapillus sp. 5 36 - Metacentric, submetacentric and telocentric Australia Rowell et al. 2002
Planipapillus sp. 6 36–38 - Metacentric, submetacentric and telocentric Australia Rowell et al. 2002
Planipapillus sp. 7 22 - Metacentric, submetacentric and telocentric Australia Rowell et al. 2002
Planipapillus sp. 8 34 - Metacentric, submetacentric and telocentric Australia Rowell et al. 2002
Ruhbergia bifalcatac Reid, 1996 (cited as Euperipatoides sp.) 30 XY - Australia Rowell et al. 1995
Tasmanipatusbarrette Ruhberg et al., 1991 40 XY - Tasmania Oliveira et al. 2018
Tetrameradenmeringosc Reid, 1996 (cited as Euperipatoides sp.) 26 XY - Australia Rowell et al. 1995
Open in a new tab

aOliveira et al. (2015) described Cerradopatus sucuriuensis as a population of Epiperipatus sp. analysed by Jeffery et al. (2012). bThe author could not exactly define the diploid number, but used a photo of a mitotic cell with 2n = 60 and found meiotic cells varying from n = 28 to n = 32. cReid (1996) assigned to different genera the species chromosomally analyzed by Rowell et al. (1995) as populations of Euperipatoides sp.

Cytogenetics provide a highly informative tool for distinguishing species of Onychophora, although it is not as well explored in Peripatidae (Oliveira et al. 2012b 2018). Epiperipatus is a genus with a monophyly that needs revision, and presents cryptic species (Oliveira et al. 2011 2012a). Thus, in this work, we described the karyotype of Epiperipatus sp. from Mato Grosso do Sul, Brazil and discuss the cytotaxonomical value of the chromosomal data to the group.

MATERIALS AND METHODS

Five specimens (4♂ and 1♀) of Epiperipatus sp. (Fig. 1) were collected at the entrance zone of three caves and surrounding areas near the Gruta Manoel Cardoso (56°43'23.85"W; 20°34'7.11"S, Bodoquena, Mato Grosso do Sul, Brazil). Only three males presented cell divisions, and were deposited in Coleção Zoológica da Universidade Federal do Mato Grosso do Sul with voucher (ZUFMS-00007).

Fig. 1.

Fig. 1.

Open in a new tab

Specimen of Epiperipatus sp. from Mato Grosso do Sul, Brazil. Photo courtesy of Dr. Paulo Robson de Souza.

We anesthetized the specimens in a chamber with ether, then dissected them by immersing individuals in a physiological solution based on Robson et al. (1966). The gonads were transferred to colchicine (Sigma Chemical CO.) solution in concentration of 0.16% (in the same physiological solution) for two hours. Then, we added an equal volume of hypotonic solution for 25 minutes and fixed the gonads in Carnoy I (3:1 methanol: acetic acid). We placed portions of gonadal tissue on a glass slide with a drop of acetic acid 60% and then, with the aid of a small metal rod, smashed the tissue to form a cell suspension before adding a few more drops of acetic acid solution to spread the material on the slide and then dry it on a metal plate at a temperature of 35 to 40°C. The slides were stained with 3% Giemsa solution (94 ml water, 3 ml phosphate buffer pH 6.8 and 3 ml Giemsa Merck-Darmstadt, Germany) for 10 minutes, except those used for FISH.

The FISH technique employed a peptidic nucleic acid (PNA) (AATCCC)3 probe (PNA Bio, Inc) complementary to the (TTAGGG)n telomeric repeats of vertebrates, labeled with Alexa fluor 488 (ThermoFisher Scientific), following the method of Genet et al. (2013), with a hybridization time of four hours at 37°C, without heat denaturing, and mounted using ProLong Diamond antifade with DAPI (ThermoFisher Scientific).

The cells were photographed with a Zeiss Axioimager D2 microscope holding a AxioCam 503 camera, using the ZEN Pro software. Chromosome morphology was determined with the free software IMAGEJ (Rasband 1997–2019) and the LEVAN plugin (Sakamoto and Zacaro 2009), according to Levan et al. (1964) and Green and Sessions (1991), using twenty-six metaphases of Epiperipatus sp.

RESULTS

Of the 47 mitotic metaphases, the three males of Epiperipatus sp. showed 2n♂ = 73 (Fig. 2A–D) (Table 2, Fig. S1). Regarding chromosomal morphology, the majority of the elements was acro/telocentric and decreased gradually in size, except for the largest chromosome of the complement, which was a single submetacentric (4.26% of the karyotype ± 0.49) and almost 50% longer than the second largest chromosome (2.91% of the karyotype ± 0.30) (Table S1). The telomeric regions of all chromosomes were hybridized with the probe to the (TTAGGG)n motif (Fig. 2B– D). The unpaired largest submetacentric chromosome has an interstitial telomeric site (ITS) in the proximal portion of the long arm (Fig. 2B, D–E; Fig. S2). No specimens presented cells during meiosis.

Fig. 2.

Fig. 2.

Open in a new tab

Chromosomes of Epiperipatus sp. (A–B) Karyotype showing 2n♂ = 73 in Giemsa staining (A) and telomeric fluorescent in situ hybridization (B). (C–D) Male mitosis used in the karyotype showed in A–B, respectively. Asterisk: large unpaired submetacentric. (E) Heteromorphic chromosome from three different metaphases. Arrow: Interstitial Telomeric Site (ITS). Scale bars = 5 μm.

Table 2.

Diploid numbers found in all analyzed metaphases of the three specimens of Epiperipatus sp.

Specimen 2n♂ = 66 2n♂ = 67 2n♂ = 68 2n♂ = 69 2n♂ = 70 2n♂ = 71 2n♂ = 72 2n♂ = 73
1 - - - 1 1 5 3 36
2 1 1 1 - - 2 - 8
3 - 1 - - - 1 - 3
Open in a new tab

DISCUSSION

The 2n♂ = 73 found in Epiperipatus sp. is the highest diploid number recorded for Onychophora up to now, with at least five more chromosomal pairs than Epiperipatus biolleyi with 2n♂ = ± 60, which had the largest diploid number previously recorded (Mora et al. 1996). Despite the fact that peripatids are the most poorly studied onychophorans, it covers the karyotypes with the highest and lowest numbers of chromosomes (2n♂ = 8 and 2n♂ = 73) for the onychophorans. Unfortunately, there is no Peripatidae phylogeny, but there is a cladogram for Peripatopsidae that uses four peripatid genera as external groups (Oliveira et al. 2018), revealing that Epiperipatus is closely related to Principapillatus, a genus that also has a high chromosome number (2n♂ = 54) (Jeffery et al. 2012; Oliveira et al. 2012b; present study). On the other hand, Eoperipatus, the genus with the lowest diploid number among onychophorans (2n♂ = 8), is basal within Peripatidae. Thus, chromosome number can be useful, along with molecular data, to reveal the evolutionary relationships in onychophoran. However, the scarcity of phylogenetic and cytogenetic data on Peripatidae reveals a weak point of this discussion.

Regarding the large submetacentric chromosome, one hypothesis is that it could be a sex chromosome. With 2n♂ = 73, an odd chromosome number, there are at least three numerical possibilities for SCS (X0, X1X2Y or XY1Y2). The presumptive occurrence of an X1X2Y or XY1Y2 SCS was already discussed for one population of Euperipatoides rowelli, 2n♂ = 33 (Peripatopsidae) (Rowell et al. 1995), but no details on chromosome size or morphology were presented for this population. The X0 SCS may have originated from an XY SCS, already found in several onychophoran species (Table 1), through heterochromatinization and deletion of the Y chromosome (Král et al. 2006). However, the presence of an ITS on the unpaired large submetacentric of Epiperipatus sp. supports an X1X2Y or XY1Y2, originated from an XY by a fusion of an autosome and a sex chromosome SCS (see Araujo et al. 2012 for a review on SCS origins in spiders). If confirmed, the putative X1X2Y or XY1Y2 SCS would have originated at least twice within Onychophora, in Peripatopsidae and in Peripatidae. An analysis of male meiosis and female mitosis in Epiperipatus sp. would allow us to distinguish among an X0 (2n♂ = 73, X0, sex univalent; 2n♀ = 74, XX), X1X2Y (2n♂ = 73, X1X2Y, sex trivalent; 2n♀ = 74, X1X1X2X2), or XY1Y2 (2n♂ = 73, XY1Y2, sex trivalent; 2n♀ = 72, XX); however, no female cell division was found.

If the large unpaired element is not a sex chromosome, then this heteromorphism may have originated through the fusion between two autosomal chromosomes. Rowell et al. (2002) observed a high chromosome number diversity in species of Planipapillus Reid, 1996, which, according to Rockman and Rowell (2002), have undergone several centric fusion events, starting from a 2n♂ = 40 ancestor karyotype with exclusively telocentric chromosomes. Therefore, the three individuals studied in this work would be heterozygous for a centric fusion, probably forming an autosomal trivalent on meiosis (the large submetacentric plus two smaller telocentrics). If this is the case, through the analysis of additional specimens, we may be able to find both homozygous individuals for the rearrangement and individuals that do not have the rearrangement.

Regardless of whether it is a sex chromosome or autosome, the results from the telomeric FISH corroborate the rearrangement, where the proximal marking found indicates a probable centric fusion. This demonstrates the importance of the telomeric FISH technique in Onychophora, because besides being informative, other techniques such as C-banding are not usually effective in this group (Rowell et al. 1995 2002; Oliveira et al. 2018).

Cryptic speciation is common in Peripatopsidae, and molecular studies in this group have found several cryptic lineages that are not distinguishable through morphological analysis. The same occurs in Peripatidae; however, it is not possible to confirm this hypothesis due to the lack of studies with non-morphological methods (Lacorte et al. 2011; Oliveira et al. 2011). Chromosome data are already used in several groups to aid in the identification of cryptic species (Dobigny et al. 2005; Řezáč et al. 2018) and Oliveira et al. (2018) comment that chromosomes can illuminate several aspects of evolution in Onychophora. Although there are few studies on Peripatidae cytogenetics, a large karyotypic diversity within the group was noted (Table 1), and in future studies, the karyotype may be fundamental in the diagnosis of Peripatidae species, as in Peripatopsidae (Rowell et al. 2002; Oliveira et al. 2012b).

CONCLUSIONS

In conclusion, the present work shows that karyotypic data can be used to aid in taxonomic studies, principally in the polyphyletic Epiperipatus, which according to Oliveira et al. (2012a), possibly contains members of Principapillatus Oliveira et al., 2013, another genus with a high chromosome number (Jeffery et al. 2012). Additionally, telomeric FISH has been shown to be important in the detection of chromosomal rearrangements that may aid in our understanding of karyotype evolution in Onychophora.

Supplementary materials

Fig. S1.

Pictures of all metaphases analyzed with score of diploid number. (download)

zoolstud-59-005-s001.pdf (12.1MB, pdf)
Fig. S2.

Pictures of telomeric fluorescent in situ hybridization in ALEXA 488, DAPI and merged. (download)

zoolstud-59-005-s002.pdf (171.5KB, pdf)
Table S1.

Relative average length of chromosomal pairs of Epiperipatus sp. (download)

zoolstud-59-005-s003.pdf (170.7KB, pdf)

Acknowledgments

The authors thank Dr. Paulo Robson de Souza of Universidade Federal de Mato Grosso do Sul (UFMS), Brazil for the photographs of the Epiperipatus sp. specimen from Mato Grosso do Sul, Brazil, and Matthijs Strietman for the language review of the manuscript. This work was supported by the BIOTA FUNDECT-CAPES under grant (SIAFEM 23519, termo de outorga 69/2014).

Footnotes

Authors’ contributions: Specimens collection and identification: LMC. Specimens dissection and slide preparations: DA LMC. Chromosome analysis: DDD DA. Manuscript writing: DDD DA LMC LHBS.

Competing interests: All authors declare that they have no conflict of interest.

Availability of data and materials: The material analyzed is available at the Coleção Zoológica da Universidade Federal do Mato Grosso do Sul.

Consent for publication: Not applicable.

Ethics approval consent to participate: Not applicable.

References

  1. Araujo D, Schneider MC, Paula-Neto E, Cella DM. 2012. Sex chromosomes and meiosis in spiders: a review. In: Swan A (ed) Meiosis-Molecular mechanisms and cytogenetic diversity. IntechOpen, Rijeka (Croatia), pp. 87–108. doi:10.5772/31612.
  2. Dobigny G, Aniskin V, Granjon L, Cornette R, Volobouev V. 2005. Recent radiation in West African Taterillus (Rodentia, Gerbillinae): the concerted role of chromosome and climatic changes. Heredity 95:358–368. doi:10.1038/sj.hdy.6800730. [DOI] [PubMed]
  3. Genet MD, Cartwright IM, Kato TA. 2013. Direct DNA and PNA probe binding to telomeric regions without classical in situ hybridization. Mol Cytogenet 6:42. doi:10.1186/1755-8166-6-42. [DOI] [PMC free article] [PubMed]
  4. Green DM, Sessions SK. 1991. Amphibian cytogenetics and evolution. Appendix I, Nomenclature for chromosomes. Academic Press, Inc., USA, pp. 431–432. doi:10.1016/C2009-0-02654-3.
  5. Instituto Chico Mendes de Conservação da Biodiversidade. 2018. Livro Vermelho da Fauna Brasileira Ameaçada de Extinção: Volume VII -Invertebrados. In: Instituto Chico Mendes de Conservação da Biodiversidade. (Org.). Livro Vermelho da Fauna Brasileira Ameaçada de Extinção. Brasília: ICMBio, p. 727.
  6. Jeffery NW, Oliveira IS, Gregory TR, Rowell DM, Mayer G. 2012. Genome size and chromosome number in velvet worms (Onychophora). Genetica 140:497–504. doi:10.1007/s10709-013-9698-5. [DOI] [PubMed]
  7. Král J, Musilová J, Št ´áhlavský F, Řezáč M, Akan Z, Edwards RL, Coyle FA, Almerje CR. 2006. Evolution of the karyotype and sex chromosome systems in basal clades of araneomorph spiders (Araneae: Araneomorphae). Chromosome Res 14:859–880. doi:10.1007/s10577-006-1095-9. [DOI] [PubMed]
  8. Lacorte GA, Oliveira IS, Fonseca CG. 2011. Phylogenetic relationships among Epiperiptus lineages (Onychophora: Peripatidae) from the Minas Gerais State, Brazil. Zootaxa 2755:57–65. doi:10.11646/zootaxa.2755.1.3.
  9. Levan A, Fredga K, Sandberg AA. 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52(2):201–220. Mora M, Herrera A, León P. 1996. The genome of Epiperipatus biolleyi (Peripatidae), a Costa Rican onychophoran. Revista de Biología Tropical 44(1):153–157.
  10. Monge-Najera J. 1995. Phylogeny, biogeography and reproductive trends in the Onychophora. Zool J Linnean Soc 114:21–60. Montgomery TH. 1900. The spermatogenesis of Peripatus (Peripatopsis) balfouri up to the formation of the spermatid. Zoologische Jahrbücher. Abteilung für Anatomie und Ontogenie der Tiere 14:277–368.
  11. New TR. 1995. Onychophora in invertebrate conservation: priorities, practice and prospects. Zool J Linnean Soc 114:77–89. doi:10.1006/zjls.1995.0017.
  12. Oliveira IS, Franke FA, Hering L, Schaffer S, Rowell DM, Weck-Heimann A, Mayer G. 2012b. Unexplored character diversity in Onychophora (velvet worms): a comparative study of three peripatid species. PLoS ONE 7(12):e51220. doi:10.1371/journal. pone.0051220. [DOI] [PMC free article] [PubMed]
  13. Oliveira IS, Hering L, Mayer G. 2020. Onychophora website. Accessed on 9 January 2020.
  14. Oliveira IS, Lacorte GA, Fonseca CG, Wieloch AH, Mayer G. 2011. Cryptic speciation in Brazilian Epiperipatus (Onychophora: Peripatidae) reveals an underestimated diversity among the peripatid velvet worms. PLoS ONE 6(6):e19973. doi:10.1371/journal.pone.0019973. [DOI] [PMC free article] [PubMed]
  15. Oliveira IS, Lacorte GA, Weck-Heimann A, Cordeiro LM, Wieloch AH, Mayer G. 2015. A new and critically endangered species and genus of Onychophora (Peripatidae) from the Brazilian savannah—a vulnerable biodiversity hotspot. Syst Biodivers 13(3):211–233. doi:10.1080/14772000.2014.985621.
  16. Oliveira IS, Lüter C, Wolf KW, Mayer G. 2014. Evolutionary changes in the integument of the onychophoran Plicatoperipatus jamaicensis (Peripatidae). Invertebr Biol 133:274–280. doi:10.1111/ivb.12063.
  17. Oliveira IS, Mayer G. 2017. A new giant egg-laying onychophoran (Peripatopsidae) reveals evolutionary and biogeographical aspects of Australian velvet worms. Org Divers Evol 17:375– 391. doi:10.1007/s13127-016-0321-3.
  18. Oliveira IS, Read VMSJ, Mayer G. 2012a. A world checklist of Onychophora (velvet worms), with notes on nomenclature and status of names. ZooKeys 211:1–70. doi:10.3897/zookeys.211.3463. [DOI] [PMC free article] [PubMed]
  19. Oliveira IS, Ruhberg H, Rowell DM, Mayer G. 2018. Revision of Tasmanian viviparous velvet worms (Onychophora: Peripatopsidae) with descriptions of two new species. Invertebr Syst 32(4):907–930. doi:10.1071/IS17096.
  20. Oliveira IS, Wieloch AH, Mayer G. 2010. Revised taxonomy and redescription of two species of the Peripatidae (Onychophora) from Brazil: a step towards consistent terminology of morphological caracters. Zootaxa 2493:16–34. doi:10.11646/zootaxa.2493.1.2.
  21. Rasband WS. 1997–2019. IMAGEJ, U.S. National Institutes of Health, Bethesda, Maryland, USA. http://imagej.nih.gov/ij/. Accessed 20 October 2019.
  22. Reid AL. 1996. Review of the Peripatopsidae (Onychophora) in Australia, with comments on peripatopsid relationships. Invertebr Taxon 10:663–936. doi:10.1071/IT9960663.
  23. Reid AL, Tait NN, Briscoe DA, Rowell DM. 1995. Morphological, cytogenetic, and allozymic variation within Cephalofovea (Onychophora: Peripatopsidae) with descriptions of three new species. Zool J Linnean Soc 114:115–138. doi:10.1006/zjls.1995.0020.
  24. Řezáč M, Arnedo MA, Opatova V, Musilová J, Rezacová V, Král J. 2018. Taxonomic revision and insights into the speciation mode of the spider Dysdera erythrina species-complex (Araneae: Dysderidae): sibling species with sympatric distributions. Invertebr Syst 32:10–54. doi:10.1071/IS16071.
  25. Robson EA, Lockwood APM, Ralph R. 1966. Composition of the blood in Onychophora. Nature 5022:533. doi:10.1038/209533a0. Rockman MV, Rowell DM. 2002. Episodic chromosomal evolution in Planipapillus (Onychophora: Peripatopsidae): a phylogenetic approach to evolutionary dynamics and speciation. Evolution 56(1):58–69. doi:10.1111/j.0014-3820.2002.tb00849. [PubMed]
  26. Rowell DM, Higgins AV, Briscoe DA, Tait NN. 1995. The use of chromosomal data in the systematics of viviparous onychophorans from Australia (Onychophora: Peripatopsidae). Zool J Linnean Soc 114:139–153. doi:10.1006/zjls.1995.0021.
  27. Rowell DM, Rockman MV, Tait NN. 2002. Extensive Robertsonian rearrangement: implications for the radiation and biogeography of Planipapillus Reid (Onychophora: Peripatopsidae). J Zool 257(2):171–179. doi:10.1017/S0952836902000778.
  28. Sampaio-Costa C, Chagas-Junior A, Baptista RLC. 2009. Brazilian species of Onychophora with notes on their taxonomy and distribution. Zoologia 26(3):553–561. doi:10.1590/S1984-46702009005000004.
  29. Sakamoto Y, Zacaro AA. 2009. LEVAN, an IMAGEJ plugin for morphological cytogenetic analysis of mitotic and meiotic chromosomes. Initial version. An open source Java plugin. http://rsbweb.nih.gov/ij/. Accessed 20 October 2019.
  30. Vítková M, Král J, Traut W, Zrzavý J, Marec F. 2005. The evolutionary origin of insect telomeric repeats, (TTAGG)N. Chromosome Res 13:145–156. doi:10.1007/s10577-005-7721-0. [DOI] [PubMed]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1.

Pictures of all metaphases analyzed with score of diploid number. (download)

zoolstud-59-005-s001.pdf (12.1MB, pdf)
Fig. S2.

Pictures of telomeric fluorescent in situ hybridization in ALEXA 488, DAPI and merged. (download)

zoolstud-59-005-s002.pdf (171.5KB, pdf)
Table S1.

Relative average length of chromosomal pairs of Epiperipatus sp. (download)

zoolstud-59-005-s003.pdf (170.7KB, pdf)

Articles from Zoological Studies are provided here courtesy of Biodiversity Research Center, Academia Sinica

RESOURCES