Chromosomal diversity in three species of Lycosa Latreille, 1804 (Araneae, Lycosidae): Inferences on diversification of diploid number and sexual chromosome systems in Lycosinae

Abstract Lycosa is one of the most speciose genera in Lycosidae, including species with different sexual chromosome systems (SCS). We carried out cytogenetic analyses in three species of Lycosa, revealing that L. erythrognatha and L. sericovittata share 2n ♂ = 22 and SCS X1X20 while L. gr. nordenskjoldi presents 2n ♂ = 19 and SCS XO, composed only of acrocentric chromosomes. All species shared pericentromeric heterochromatin. Nonetheless, one specimen of L. sericovittata carried two chromosomes with terminal heterochromatin and L. gr. nordenskjoldi showed four chromosomes with interstitial heterochromatin plus another chromosome with terminal C-bands. The pericentromeric heterochromatin of all species as well as the terminal heterochromatic blocks in L. sericovittata were CMA3 + . The 18S rDNA sites varied in number and type of bearing chromosomes both at inter and intrapopulational levels, with the highest variation in L. gr. nordenskjoldi. These differences may be related to gene dispersal due to the influence of transposition elements and translocation events. Despite these variations, all species shared ribosomal sites in pair 5. This study demonstrated intra and interspecific chromosomal variability of Lycosa, suggesting that chromosomal rearrangements are related to the diversification of diploid number and SCS in this group of spiders.

Subsequently, Forman et al. (2013) identified in Wadicosa fidelis (O. Pickard-Cambridge, 1872), by silver nitrate staining and fluorescent in situ hybridization (FISH) with 18S rDNA probes, three and seven to ten NOR sites, respectively.
Based on these data, the goal of this study was to extend the chromosomal information in this group of spiders by including refined cytogenetic analyzes. Therefore, different chromosome banding techniques were applied to three Lycosa species from Paraná state, Brazil, to investigate karyotypic variability, presence of different SCS, heterochromatin distribution patterns and chromosome mapping of 18S rDNA sites. Based on a comparative approach, we discuss some of the mechanisms that could account for the karyotype differentiation in Lycosinae species.

Material and Methods
Cytogenetic analyses were performed in three species of Lycosa, collected in five locations along the state of Paraná (Table 1). The specimens were deposited in the arachnological collection of the Laboratory of Zoological Collections at the Butantan Institute (IBSP, curator A.D. Brescovit) in São Paulo/SP, Brazil. Chromosomal preparations were obtained according to Araujo et al. (2008), using young and adult spider testicles. The slides were stained with Giemsa 3%, and approximately 30 mitotic and meiotic cells from each individual were analyzed to determine the diploid number. The morphology and chromosome measurements were performed on 10 mitotic metaphases in the Image J program  with the use of the plugin LEVAN (Sakamoto and Zacaro, 2009), according to the methodology described by Levan et al. (1964). The slides were submitted to C banding according to Sumner (1972), modified by Lui et al. (2012). The staining with base-specific fluorochromes chromomycin A 3 (CMA 3 ) and 4,6'-diamidino-2'phenylindol (DAPI) was carried out according to Schweizer (1980). Fluorescent in situ hybridization (FISH) was performed according to Schwarzacher and Heslop-Harrison (2000). The 18S rDNA probes were obtained from Ctenus ornatus (Keyserling, 1877) by Rincão et al. (2017), labeled with biotin by using the Biotin-Nick Translation Mix (Roche) kit and detected with Avidin-FITC (Invitrogen). Finally, the FISH slides were analyzed using an epifluorescence microscope (Leica DM2000), equipped with digital camera Moticam Pro 282B. The images were captured using the program Motic Images Advanced, version 3.2.

Results
Lycosa erythrognatha and L. sericovittata presented 2n♂= 22 and SCS X 1 X 2 0 ( Figure 1A, B, respectively), while L. gr. nordenskjoldi presented 2n♂= 19 and SCS X0 ( Figure  1C); The karyotypes of the three species are entirely composed of acrocentric chromosomes. However, each species showed different sizes of the sex chromosomes: X 1 and X 2 elements are the two largest chromosomes (6.96 -6.74 µm respectively) in L. erythrognatha, where the other chromosomes vary from 5.65 -3.18 µm; in L. sericovittata X 1 is a medium-sized chromosome of 3.82 µm, significantly distancing itself from the first and largest pair (6.30 µm) and X 2 is one of the smallest elements (2.69 µm), the other pairs vary 6.05 -2.94 µm. On the other hand, the X chromosome is the smallest element (3.33 µm) in the karyotype of L.gr. nordenskjoldi, and the other chromosomes vary from 6.48 -3.88 µm (Figure 1).
In meiotic cells of males from the three species, the sex chromosomes are easily visible in the pachytene nucleus due to their high condensation, being frequently observed as single mass of positive heteropycnosis (Figure 2A, E, I). The diplotene cells in L. erythrognatha and L. sericovittata ( Figure  2B, F, respectively) showed 10 autosomal bivalents and two sexual univalents (10II+X 1 X 2 ), whereas L. gr. nordenskjoldi ( Figure 2J) presented 9 autosomal bivalents and one sexual univalent (9II+X), with predominance of terminal chiasmata in the three species. In diakinesis, sexual univalent arranged side by side or very close to each other were observed in both species with X 1 X 2 0 SCS ( Figure 2C, G).   Metaphase II cells showed 10 and 12 chromosomes in L. erythrognatha ( Figure 2D) and L. sericovittata ( Figure 2H), with the sexual univalents segregation to the same pole, confirming the X 1 X 2 0 SCS. In L. gr. nordenskjoldi ( Figure 2L) 10 and 9 chromosomes were detected in cells during metaphase II, thereby confirming the X0 SCS.
The C-banding applied to testicular cells of the three species ( Figure 3A, B, C), showed pericentromeric heterochromatin in all chromosomes. Moreover, L. sericovittata also showed a chromosome pair with terminal C-bands ( Figure 3B) while L. gr. nordenskjoldi presented some interstitial C-bands regions ( Figure 3C).

Cavenagh et al. 4
The base-specific fluorochrome staining revealed interspecific differences: Lycosa erythrognatha and L. gr. nordenskjoldi ( Figure 3D, F, respectively) presented CMA 3 + pericentromeric signals in all chromosomes, coinciding with heterochromatic regions in the former. In addition to the pericentromeric CMA 3 + regions, L. sericovittata also presented GC-rich sites at terminal regions of two chromosomes ( Figure  3E). No DAPI + signals (AT-rich sites) were detected in the analyzed species (data not shown).
The FISH experiments revealed four 18S rDNA sites in L. erythrognatha, with interpopulation variation of 18S-bearing pairs. Therefore, these ribosomal cistrons were located in pairs 5 and 9 of three individuals from Mata dos Godoy State Park (PEMG) and the four specimens from the State University of Londrina (UEL) ( Figure 4A), while 10 individuals from Superagui National Park (PNS) and three from Iguaçu National Park (PNI) presented positive signals in pairs 2 and 5 ( Figure 4B). In L. sericovittata, FISH also identified four 18S rDNA sites, at the terminal region of pairs 5 and 9 of the two individuals analyzed ( Figure 4C).
In addition, inter and intrapopulational variation in the number of 18S rDNA was observed in L. gr. nordenskjoldi, ranging from four, six and seven 18S signals. Similarly, this species also presented variation in the pairs bearing ribosomal sites, as follows: two individuals from Ilha Grande National Park (PNIG) showed four 18S rDNA sites at terminal region of pairs 5 and 9 ( Figure 5A) while two individuals from the same locality presented six sites in pairs 2, 3 and 5 ( Figure 5B). On the other hand, the three samples from PNS were characterized by seven 18S rDNA sites at terminal region of pairs 1, 5 and 8, and on a single chromosome from pair 3 ( Figure 5C).
It should be pointed out that species of Lycosa show considerable levels of chromosomal variation in spite of the low number of species analyzed so far. This feature and the fact that this genus is recognized as a polyphyletic group composed of many species, jeopardizes reliable estimates about the ancestral diploid number in Lycosa and their karyoevolutionary relationships.

Patterns of heterochromatin distribution
The three species analyzed shared the common pattern of heterochromatin distribution described by Chemisquy et al. (2008), including C-bands and GC-rich segments at pericentromeric regions. However, variations were found in these species, such as the presence of GC-rich terminal sites in a specimen of L. sericovittata. Moreover, L. gr. nordenskjoldi was characterized by heterogeneity of heterochromatin distribution due to the occurrence of pericentromeric, interstitial and terminal C-bands while GC-rich sequences were restricted to the pericentromeric region. Therefore, two additional patterns of C-banding were identified in this study: (1) the presence of terminal GC-rich heterochromatin segments; and (2) interstitial heterochromatin with no signs of GC or AT richness (CMA 3 -/DAPI -). The distribution of heterochromatic blocks at pericentromeric regions, had been considered as distinctive feature within Lycosidae, as supported by the data reported by Chemisquy et al. (2008) and the present results. Nevertheless, our data demonstrated novel patterns of heterochromatin distribution in this group of spiders in which L. gr. nordenskjoldi stands out by the high dispersal of heterochromatin segments. A comparative analysis between these results and the putative ancestor pattern of heterochromatin distribution in Lycosa suggests that paracentric inversions or dispersal of repetitive sequences could be related to the C-banding pattern described in L. gr. nordenskjoldi.
The variability in the data obtained by C-banding and fluorochrome staining, particularly in Lycosa gr. nordenskjoldi, in addition to the diploid number, confirms that traditional chromosomal markers allow differentiating congeneric species, at least in comparison with data described in literature so far.

Interpopulation chromosomal variability of 18S rDNA sites
Despite the presence of two 18S rDNA-bearing pairs in L. erythrognatha, this species showed interpopulation variation in the position of these sites in different chromosomes in the karyotypes. The Superagui National Park (PNS), located on the northern coast of the state of Paraná, is a region of islands and mangroves with a more tropical climate, similar to that of the Iguaçu National Park (PNI) in the western boundary of Paraná, which includes one of the largest conserved areas of Atlantic Forest in Brazil. The populations of L. erythrognatha from both regions (PNS and PNI) exhibited a different karyotypic pattern in relation to those from the northern region (PEMG and UEL), a region of dry climate and characterized by semideciduous seasonal forest vegetation. In spite of the geographic distance between these locations (PNS and PNI), which is about 560 km, we infer that adaptive processes in these populations should be comparable to each other because they share similar habitats. Analogously, the environmental differences among the four populations should impose differential selective pressure, thereby determining distinct evolutionary pathways.
This interpopulation variation in 18S rDNA-bearing pairs may be related to gene dispersal via transpositions or translocations, as suggested by Cabral de Melo et al. (2011) in a study with beetles (Scarabaeinae). These authors point out that in the absence of significant karyotypic changes (e.g., increase or decrease in diploid numbers), the ribosomal sites can disperse and vary as a result of successive amplification processes of these cistrons, particularly when located at distal portion of chromosomes inasmuch as these regions are considered highly dynamic, thus favoring the dispersal of rDNA copies throughout the genome.
The distribution of ribosomal sites in L. gr. nordenskjoldi showed both inter and intrapopulation variation. The variability between the two populations (PNS and PNIG) of this species also can be related to their habitat. Despite being similar to each other, the evolutionary pressure can act in different ways on populations from distinct species, eventually resulting in independent accumulation of chromosomal rearrangements in locally adapted individuals, as previously reported in Wadicosa fidelis (Forman et al., 2013) and in harvestmen species (Opiliones, Phalangiidae) (Šťáhlavský et al., 2018).
On the other hand, the presence of 18S rDNA sites in pairs 5 and 9 was shared by the three species, with pair 5 observed in all populations, despite the variability in location and number of ribosomal cistrons. Apparently, this would be a conserved trait in these species what remains to be confirmed by further studies, since this is the first report based on FISH experiments in Lycosa.

Chromosomal diversification within Lycosinae
Recent phylogenetic inferences (Piacentini and Ramiréz, 2019), revealed that most species of Lycosidae from south America represent undescribed genera, what should explain the karyotypic diversity observed in literature and in the present work. Furthermore, the South American species usually present lower diploid numbers than Eurasian representatives (Araujo et al., 2021).
In addition, the phylogenetic reconstruction presented by Piacentini and Ramiréz (2019), Lycosinae encompasses species from North and South American, as well as the genus Hogna and Eurasian species of Lycosa. The latter, along with the outgroup Pardosinae, has a predominance of species with 2n = 28, X 1 X 2 0 (Araujo et al., 2021), which can be suggested as the ancestral diploid number of the Lycosinae subfamily.
Analyzing the data presented in Araujo et al. (2021), these unusual diploid numbers were determined by alterations in the SCS, related to increases or decreases in the number of chromosomes with the consequent evolution of new SCS. As mentioned earlier, X 1 X 2 0 SCS is regarded as an ancestral condition for several groups of spiders, including Lycosoidea (Dolejš et al., 2011;Araujo et al., 2015). Thus, other SCS systems should be considered as derived features. Some studies, including those by Král et al. (2006) and Araujo et al. (2012Araujo et al. ( , 2014, have previously demonstrated that X 1 X 2 X 3 0 and X 1 X 2 0 SCS coexist within a single genus or, even, in the same species (Araujo et al., 2014;Rincão et al., 2020).
The origin of the above mentioned SCSs in Entelegynae was hypothesized by several authors as follows: 1) by fusions or fissions in the sex chromosomes during the conversion of X 1 X 2 0 to X 1 X 2 X 3 0 system and vice versa, and during the conversion of X 1 X 2 0 to a single X0 system (Pätau, 1948;Postiglioni and Brum-Zorrilla, 1981;Parida and Sharma, 1986); 2) by the formation of a supernumerary element during the conversion of X 1 X 2 0 to X 1 X 2 X 3 0 system (Bole-Gowda, 1952); and 3) by fusions between sex and autosomal chromosomes, especially during the conversion of X 1 X 2 0 to X 1 X 2 Y system (Král et al., 2006). Despite this great variability in SCS, Entelegynae spiders share two notable characteristics, which are the predominance of acrocentric chromosomes and the occurrence of X 1 X 2 0 SCS (Král et al., 2006;Araujo et al., 2014), which is observed in Lycosinae.
One of the most cited chromosomal rearrangements is the fusion between autosomes and sex chromosomes, as proposed by Hackman (1948), resulting in metacentric elements, usually followed by pericentric inversions or partial deletion Chatterjee, 1989, 1992). Another event often hypothesized within this context would be the in tandem fusion, resulting in the origin of acrocentric chromosomes (Pekár and Král, 2001). When changes in diploid number take place without modifications in the X 1 X 2 0 SCS, rearrangements such as single translocation or in tandem fusion among autosomal chromosomes are inferred, thus maintaining the acrocentric/ telocentric chromosomal set. Such event might have caused the differentiation of karyotypes with 2n♂ = 26, 24, 22 and 18. These diploid numbers are reported, for example, in Gladicosa pulchra (Keyserling, 1877); Lycosa madani Pocock, 1901;Schizocosa malitiosa (Tullgren, 1905); and Lycosa tarantula (Linnaeus, 1758), respectively.
On the other hand, when changes in both diploid numbers and SCS occur, the even diploid number is modified into an odd chromosome number. In this case, an X 1 X 2 0 SCS originates the novel and less frequent systems: X0, X 1 X 2 X 3 0 and X 1 X 2 Y, present in some species of Lycosa (South America) associated with distinctive morphology of sex chromosomes. Accordingly, the X 1 X 2 X 3 0 system could have arisen from the insertion of a supernumerary chromosome in the former X 1 X 2 0 SCS (Bole- Gowda, 1952) or from chromosomal nondisjunction (Postiglioni and Brum-Zorrilla, 1981;Datta and Chatterjee, 1988). Additionally, the X 1 X 2 Y SCS could emerge after a translocation between sex and autosomal chromosomes (Silva et al., 2002;Rowell, 2004;Král et al., 2006Král et al., , 2007.
Therefore, the presence of lower diploid numbers and unusual SCS is likely to derive from karyotypes with similar 2n values instead of a series of fusions in former karyotypes with 2n ♂ = 28. For example, the occurrence of 2n♂ = 23, X 1 X 2 X 3 0 should rather evolve from 2n ♂ = 22, X 1 X 2 0 by the formation of a supernumerary element than through multiple fusion/fission events.
In conclusion, this study demonstrated a wide variation in chromosomal features among and within the three species of Lycosa, as evidenced by the differences in both number and location of 18S rDNA sites and heterochromatic blocks, especially in the species complex Lycosa gr. nordenskjoldi. The data also showed that genomes have undergone chromosomal breaks and translocation/chromosome fusions, which account for the differentiation of diploid numbers and sex chromosomes system in species of Lycosinae.