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Small pholcids (Araneae: Synspermiata) with big surprises: the lowest diploid number in spiders with monocentric chromosomes.

Knowledge regarding spider cytogenetics has grown considerably in recent years; however, less than 2% of the ca. 47,000 described species are karyotyped so far (Araujo et al. 2017; World Spider Catalog 2017). In spite of this, there is a huge variation in the diploid number, from 2n? = 7, in the dysderid Dasumia carpatica (Kulczynski, 1882) and the segestriid Ariadna lateralis Karsch, 1881 (Suzuki 1950, 1954; Korinkova & Kral 2013), to 2n[male] = 128, in the ctenizid Cycloscoma siamensis Schwendinger, 2005 (Kral et al. 2013). In general, lower diploid numbers are found in araneomorphs whilst higher diploid numbers prevail in non-araneomorphs (see Araujo et al. 2017).

The Pholcidae is the ninth and second most diverse family among Araneae and Synspermiata spiders, respectively, with about 1600 species and 80 genera (World Spider Catalog 2017). However, only 19 species belonging to eight genera were cytogenetically studied, with the diploid number varying from 2n[male] = 15 to 2n[male] = 32. The X0 sex chromosome system (SCS) predominates within the pholcids, occurring in 13 species, followed by the [X.sub.1][X.sub.2]0, [X.sub.1][X.sub.2]Y and XY systems. Similar to most Synspermiata spiders, the chromosomes are, in general, biarmed (see Araujo et al. 2017).

Huber (2011), taking into account several phylogenetic hypotheses, updated a classification of pholcids in five subfamilies: Ninetinae, Arteminae, Modisiminae, Smeringopinae and Pholcinae. This classification was latter corroborated by molecular phylogenies (Dimitrov et al. 2013). Among these subfamilies, only Ninetinae has no cytogenetic data (see Araujo et al. 2017), but many gaps remain, even in the other subfamilies, which are underrepresented in this type of study. For Neotropical pholcids, there are cytogenetics data published for only three native species, all of them belonging to the genus Mesabolivar Gonzalez-Sponga, 1998 (see Araujo et al. 2017): M. brasiliensis (Moenkhaus, 1898), M. cyaneotaeniatus (Keyserling, 1891) and M. luteus (Keyserling, 1891). Mesabolivar is a highly species-rich genus with 64 described species and dozens of hitherto undescribed species available in museums, all of them exclusively found in South America (Huber 2015; World Spider Catalog 2017; L.S. Carvalho, pers. obs.). Previous chromosomal studies in the Mesabolivar species revealed 2n[male] = 17, X0 and 2n[male] = 15, X0, with meta/submetacentric chromosomes (Araujo et al. 2005a; Ramalho et al. 2008).

Here, we provide chromosomal data for five Neotropical pholcid species: three Modisiminae--Mesabolivar spinulosus (Mello-Leitao, 1939), Mesabolivar togatus (Keyserling, 1891) and Carapoia sp.--and two Pholcinae--Micropholcus piaui Huber, Carvalho & Benjamin, 2014 and Micropholcus ubajara Huber, Carvalho & Benjamin, 2014. The genus Micropholcus Deeleman-Reinhold & Prinsen, 1987 (Pholcinae) possesses 16 species, mainly from South America and the Caribbean region (Huber et al. 2014). The only species cytogenetically characterized of this genus is its type-species, the synanthropic pantropical Micropholcus fauroti (Simon, 1887), that exhibits 2n[male] = 17, X0, and metacentric chromosomes in a Brazilian population (Araujo et al. 2005a). Carapoia Gonzalez-Sponga, 1998 (Modisiminae) was never analyzed from the cytogenetic point of view.

METHODS

A total of 38 specimens from the Brazilian fauna were analyzed: 11 individuals (10[male] and 1[female])of Mesabolivar spinulosus, from Fazenda Bonito (05[degrees]14'S, 41[degrees]41'W), municipality of Castelo do Piaui, state of Piaui; two males of Mesabolivar togatus, from the municipality of Vicosa (20[degrees]45T4"S, 42[degrees]52'55"W), state of Minas Gerais; seven specimens (6? and 19) of an undescribed species of the genus Carapoia, from Parque Nacional de Ubajara (03[degrees]49'S, 40[degrees]59'W), municipality of Ubajara, state of Ceara;14 topotype specimens of Micropholcus piaui (10[male] and 4[female]), from Parque Municipal da Pedra do Castelo (05[degrees]12'S, 41[degrees]41'W), municipality of Castelo do Piaui, state of Piaui; and four topotype individuals (2[male] and 2[female])of Micropholcus ubajara, from Gruta do Morcego Branco, Parque Nacional de Ubajara (03[degrees]49'S, 40[degrees]59'W), municipality of Ubajara, state of Ceara. After the gonads extraction, the specimens were deposited at Laboratorio Especial de Colecoes Zoologicas, Instituto Butantan (IBSP, curator A.D. Brescovit), Sao Paulo, state of Sao Paulo, and Colecao de Historia Natural, da Universidade Federal do Piaui (CHNUFPI, curator E.F.B. Lima), Floriano, state of Piaui, Brazil. The collecting permit was issued by the Instituto Chico Mendes de Conservacao da Biodiversidade -ICMBio, through the Sistema de Autorizacao e Informacao em Biodiversidade - SISBIO (#39233-1).

The gonads were dissected out and processed for chromosomal preparations, according to Araujo et al. (2005b). The cells were photographed in an Olympus BX51 microscope, using the DP software, or in a Zeiss Axio Imager A2, using the Axio Vision software. In both cases, the total magnification was 1600x. The chromosomal morphology was identified following the proposal of Levan et al. (1964).

RESULTS

All five species analyzed here present a SCS of the X0/XX type, which was confirmed due to the difference of one chromosome between male and female mitotic metaphase cells (except in Mesabolivar togatus, in which females were not analyzed), presence of only one chromosomal univalent in male diplotene, and occurrence of the X chromosome in only one pole in metaphase II cells. In the five species, the X chromosome is the largest or nearly the largest element of the karyotype, and all chromosomes are biarmed.

Mitotic metaphase nuclei of Mesabolivar spinulosus possess 2n[male] = 17 and 2n[female] = 18 (Fig. 1A), and spermatogonia of Mesabolivar togatus has 2n[male] = 17 (Fig. 1B). Male diplotene nuclei of both species revealed eight autosomal bivalents with one or two interstitial or terminal chiasmata, and one sex univalent, which was easily recognized due to its length and configuration (Fig. 2A & B). Male metaphase II cells of two Mesabolivar species present n = 8or n= 8 + X. The chromosome morphology of M. spinulosus is metacentric (pairs 1, 2, 4 and X), subtelocentric (pairs 3 and 8), and submetacentric (pairs 5, [+ or -] and 7). Mesabolivar togatus presents chromosomes with metacentric (pairs 1, 3-6 and X) and submetacentric (2, 7 and 8) morphology (Fig. 1A & B).

Mitotic metaphase cells of Carapoia sp. reveal 2n[male] = 15 (Fig. 1C) and 2n[female] = 16. Male diplotene nuclei show seven autosomal bivalents with one or two terminal or interstitial chiasmata, and one sex univalent (Fig. 2C). Male metaphase II cells possess n = 7or n= 7 + X (Fig. 2D). The chromosomal morphology is exclusively metacentric (Figs. 1C, 2D).

Mitotic metaphase cells of Micropholcus piaui and Micropholcus ubajara (Pholcinae) present 2n[male] = 9 (Fig. 1D & E) and 2n[female] = 10. Male diplotene/metaphase I nuclei showed four autosomal bivalents, with one terminal or interstitial chiasma in M. piaui, and one or two terminal or interstitial chiasmata in M. ubajara. The sex chromosome is univalent, identified due to its positive heteropycnosis (Fig. 2E & F). In both species, male metaphase II nuclei reveal n = 4or n= 4 + X (Fig. 2G & H). In these species, the chromosomal morphology is exclusively metacentric, except in the submetacentric pair 2 of M. ubajara (Fig. 1D & E).

DISCUSSION

The karyotype characteristics (X0 SCS, a large biarmed X chromosome, and biarmed autosomes) observed in the five species studied here are the most common among the Pholcidae (see Araujo et al. 2017). Within Mesabolivar (Modisiminae), the 2n[male] = 17, X0 was previously found in M. brasiliensis and M. cyaneotaeniatus (Ramalho et al. 2008). However, the Pholcidae molecular phylogenetic analyses carried out by Dimitrov et al. (2013) did not recover the monophyly of Mesabolivar representatives analyzed.

The phylogenetic position of M. spinulosus and M. togatus within Mesabolivar is unknown as these species were not included in the only available phylogenetic analysis of the Pholcidae (Dimitrov et al. 2013). However, these species, along with M. brasiliensis, M. cyaneotaeniatus, (all with 2n[male] = 17), and others, forms a group of Mesabolivar species with a ''distinctively curved procursus'', called ''southern group'' (Huber 2000). Moreover, these four species of Mesabolivar with 2n? = 17 present a mixture of meta/submeta/subtelocentrics, or at least meta/submetacentrics, while M. luteus presents exclusively metacentric chromosomes, reinforcing that this last species is not closely related to the species with 2n[male] = 17.

Carapoia is the second genus of Modisiminae chromosomally studied, and the results shown herein suggest a close phylogenetic relationship between M. luteus and Carapoia species based on the chromosome number and morphology. In fact, the generic allocation of M. luteus has a long history of uncertainty (see Huber 2000, 2005; Astrin et al. 2007), and the new chromosomal data corroborates the most recent molecular data (Dimitrov et al. 2013) and the female morphology (Huber 2000), pointing towards its position in the genus Carapoia.

Interestingly, within pholcids, the karyotype 2n [male] = 15, X0, such as herein observed in Carapoia sp., was previously found only in the modisimine M. luteus (Araujo et al. 2005a), and in representatives of the arteminae genus Physocyclus Simon, 1893 (Cokendolpher & Brown 1985; Cokendolpher 1989; Oliveira et al. 2007; Golding & Paliulis 2011). According to the phylogenetic hypothesis of Dimitrov et al. (2013), Arteminae and Modisiminae are sister clades, but the considerably lack of cytogenetic data prevents any further comparison.

The phylogenetic analysis of Huber et al. (2014) recovered the monophyly of Micropholcus species, but with some uncertain relationship between the South American clade, the Caribbean clade and the Micropholcus type-species, the pantropical M. fauroti. The chromosomal data obtained in M. piaui and M. ubajara (2n[male] = 9, X0), both Brazilian species, strongly differ from that of M. fauroti (2n[male] = 17, X0) (Araujo et al. 2005a). Huber et al. (2014) placed the New World Micropholcus species in this genus mostly because they were not able to find any morphological synapomorphies that would have supported the establishment of a new genus. The lower diploid number observed in M. piaui and M. ubajara is the first morphological putative synapomorphy that would support a new genus including the New World Micropholcus species. Analyses of Caribbean Micropholcus, unknown from the cytogenetic point of view, reveals to be important to verify whether a low diploid number is also present in this clade of Micropholcus.

The number of autosomes in M. piaui and M. ubajara (eight) is exactly half of that found in M. fauroti (sixteen). Thus, one plausible hypothesis for the origin of the karyotype observed in M. piaui and M. ubajara is the occurrence of an ''all or nothing'' fusion event, in which all autosomes from the 2n[male] = 17 = 16 + X0 have fused with each other. This type of mechanism has been proposed to explain the origin of the karyotype of some spider species (Suzuki 1954; Rowell 1990; Stavale et al. 2011). As in all these Micropholcus the chromosomes are biarmed, it is possible that tandem fusions, instead of centric fusion, were implied in the process. This rearrangement was preceded or followed by pericentric inversions, in a way that keep both chromosome arms of each chromosome of similar sizes. The proposition that tandem fusions, instead of centric fusions, occurred in spiders that experienced a reduction in chromosome number and maintenance of chromosomal morphology was already postulated by Kral et al. (2006), but referring to acrocentric karyotypes. In the case of M. piaui and M. ubajara, it is possible that the putative dicentric chromosomes, products of the fusion, undergo an inactivation of one centromere, a process already well documented in several organisms (Stimpson et al. 2012).

The 2n[male] = 9, observed in M. piaui and M. ubajara is the lowest diploid number recorded for spiders with monocentric chromosomes. Low diploid numbers are found in Ariadna lateralis (Segestriidae) and Dasumia carpatica (2n? = 7), Dysdera crocata C.L Koch, 1838 (2n[male] = 8) (Dysderidae), and Ariadna bosenbergi Keyserling, 1877 (2n[female] = 8) (Suzuki 1950, 1954; Diaz et al. 2010; Korinkova & Kral 2013), however, these species possess holocentric chromosomes.

In conclusion, the present cytogenetical data support a separate evolutionary position of Mesabolivar luteus in relation to the remaining Mesabolivar representatives and show that, within the Pholcidae, the 2n[male] = 15, X0 is present only in the sister subfamilies Modisiminae and Arteminae. Moreover, we could show the lowest diploid number in spiders with monocentric chromosomes, 2n = 9. Thus, our findings raise questions on the evolution of the low diploid number in the genus Micropholcus, requiring further investigation in Caribbean species or hitherto undescribed Old World species.

ACKNOWLEDGMENTS

This work is part of LSC's Ph.D. thesis and part of the Programa de Pesquisas em Biodiversidade do Semiarido (CNPq 558317/2009-0 and 457471/2012-3). This research was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, FAPESP (2011/21643-1 and 2012/10679-8). We are grateful to Bernhard A. Huber and anonymous referees for their critical reading of the manuscript.

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Manuscript received 5 May 2017, revised 6 October 2017.

Rafael Lucena Lomazi (1), Douglas Araujo (2), Leonardo Sousa Carvalho (3,4) and Marielle Cristina Schneider (1): (1) Universidade Federal de Sao Paulo, UNIFESP, Departamento de Ciencias Biologicas, Av. Prof. Artur Riedel, 275, 09972-270, Diadema, Sao Paulo, Brazil. E-mail: marielle.unifesp@gmail.com; (2) Universidade Federal de Mato Grosso do Sul, UFMS, Setor de Biologia Geral, Instituto de Biociencias, Cidade Universitairia, Bairro Universitairio, 79070-900, Campo Grande, Mato Grosso do Sul, Brazil. (3) Universidade Federal do Piaui, UFPI, Campus Amilcar Ferreira Sobral, Departamento de Ciencias Biologicas, BR 343, km 3.5, Bairro Meladao, 64800-000, Floriano, Piaui, Brazil. (4) Pos-Graduacao em Zoologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
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Author:Lomazi, Rafael Lucena; Araujo, Douglas; Carvalho, Leonardo Sousa; Schneider, Marielle Cristina
Publication:The Journal of Arachnology
Article Type:Report
Geographic Code:3BRAZ
Date:Jan 1, 2018
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