Integrative Taxonomy Approach Reveals Cryptic Diversity within the Phoretic Pseudoscorpion Genus Lamprochernes (Pseudoscorpiones: Chernetidae).

Pseudoscorpions represent an ancient, but homogeneous group of arachnids. The genus Lamprochernes comprises several morphologically similar species with wide and overlapping distributions. We implemented an integrative approach combining molecular barcoding (cox1), with cytogenetic and morphological analyses in order to assess species boundaries in European Lamprochernes populations. The results suggest ancient origins of Lamprochernes species accompanied by morphological stasis within the genus. Our integrative approach delimited three nominal Lamprochernes species and one cryptic lineage Lamprochernes abditus sp. nov. Despite its Oligocene origin, L. abditus sp. nov. can be distinguished from its closest relative only by molecular and cytogenetic differences, or alternatively, by a complex multivariate morphometric analysis involving other Lamprochernes species. The population structure and common haplotype sharing across geographically distant populations in most Lamprochernes species suggest that a phoretic manner of dispersal is efficient in this group.

Free-living Trichomonads are Unexpectedly Diverse.

The vast majority of the more than 450 described species of Parabasalia are intestinal symbionts or parasites of animals. This endobiotic life-history is presumably ancestral although the root of Parabasalia still needs to be robustly established. The half-dozen putatively free-living species thus far described are likely independently derived from endobiotic ancestors and represent the most neglected ecological group of parabasalids. Thus, we isolated and cultivated 45 free-living strains of Parabasalia obtained from a wide variety of anoxic sediments to conduct detailed morphological and SSU rRNA gene phylogenetic analyses. Sixteen species of trichomonads were recovered. Among them, we described seven new species, three new genera, two new families, and one new order. Most of the newly described species were more or less closely related to members of already described genera. However, we uncovered a new deep-branching lineage without affinity to any currently known group of Parabasalia. The newly discovered free-living parabasalids will be key taxa in comparative analyses aimed at rooting the entire lineage and deciphering the evolutionary innovations involved in transitioning between endobiotic and free-living habitats.

Molecular data do not support the traditional morphology-based groupings in the scorpion family Buthidae (Arachnida: Scorpiones).

The family Buthidae represents an early-diverging and most species-rich lineage of extant scorpions, but its internal phylogenetic relationships are still poorly understood. The family is traditionally divided into six morpho-groups; however, the monophyly of some of them remains unclear. We combined multilocus sequence data with extensive taxon sampling to reconstruct the phylogenetic relationships among Buthidae and assess the validity of the morphology-based groupings. We recovered a monophyletic Buthus group as a sister clade to all the remaining Buthidae. We also found support for the monophyly of the Tityus group, but the remaining morpho-groups were recovered as para-/polyphyletic. Our results also suggest that some genera are in need of a taxonomic revision.

Karyotype differentiation and male meiosis in European clades of the spider genus Pholcus (Araneae, Pholcidae).

Haplogyne araneomorphs are a diverse spider clade. Their karyotypes are usually predominated by biarmed (i.e., metacentric and submetacentric) chromosomes and have a specific sex chromosome system, X1X2Y. These features are probably ancestral for haplogynes. Nucleolus organizer regions (NORs) spread frequently from autosomes to sex chromosomes in these spiders. This study focuses on pholcids (Pholcidae), a highly diverse haplogyne family. Despite considerable recent progress in pholcid cytogenetics, knowledge on many clades remains insufficient including the most species-rich pholcid genus, Pholcus Walckenaer, 1805. To characterize the karyotype differentiation of Pholcus in Europe, we compared karyotypes, sex chromosomes, NORs, and male meiosis of seven species [P.alticeps Spassky, 1932; P.creticus Senglet, 1971; P.dentatus Wunderlich, 1995; P.fuerteventurensis Wunderlich, 1992; P.phalangioides (Fuesslin, 1775); P.opilionoides (Schrank, 1781); P.silvai Wunderlich, 1995] representing the dominant species groups in this region. The species studied show several features ancestral for Pholcus, namely the 2n♂ = 25, the X1X2Y system, and a karyotype predominated by biarmed chromosomes. Most taxa have a large acrocentric NOR-bearing pair, which evolved from a biarmed pair by a pericentric inversion. In some lineages, the acrocentric pair reverted to biarmed. Closely related species often differ in the morphology of some chromosome pairs, probably resulting from pericentric inversions and/or translocations. Such rearrangements have been implicated in the formation of reproductive barriers. While the X1 and Y chromosomes retain their ancestral metacentric morphology, the X2 chromosome shows a derived (acrocentric or subtelocentric) morphology. Pairing of this element is usually modified during male meiosis. NOR patterns are very diverse. The ancestral karyotype of Pholcus contained five or six terminal NORs including three X chromosome-linked loci. The number of NORs has been frequently reduced during evolution. In the Macaronesian clade, there is only a single NOR-bearing pair. Sex chromosome-linked NORs are lost in Madeiran species and in P.creticus. Our study revealed two cytotypes in the synanthropic species P.phalangioides (Madeiran and Czech), which differ by their NOR pattern and chromosome morphology. In the Czech cytotype, the large acrocentric pair was transformed into a biarmed pair by pericentric inversion.

Insights into the Karyotype Evolution of Charinidae, the Early-Diverging Clade of Whip Spiders (Arachnida: Amblypygi).

Whip spiders (Amblypygi) represent an ancient order of tetrapulmonate arachnids with a low diversity. Their cytogenetic data are confined to only a few reports. Here, we analyzed the family Charinidae, a lineage almost at the base of the amblypygids, providing an insight into the ancestral traits and basic trajectories of amblypygid karyotype evolution. We performed Giemsa staining, selected banding techniques, and detected 18S ribosomal DNA and telomeric repeats by fluorescence in situ hybridization in four Charinus and five Sarax species. Both genera exhibit a wide range of diploid chromosome numbers (2n = 42-76 and 22-74 for Charinus and Sarax, respectively). The 2n reduction was accompanied by an increase of proportion of biarmed elements. We further revealed a single NOR site (probably an ancestral condition for charinids), the presence of a (TTAGG)n telomeric motif localized mostly at the chromosome ends, and an absence of heteromorphic sex chromosomes. Our data collectively suggest a high pace of karyotype repatterning in amblypygids, with probably a high ancestral 2n and its subsequent gradual reduction by fusions, and the action of pericentric inversions, similarly to what has been proposed for neoamblypygids. The possible contribution of fissions to charinid karyotype repatterning, however, cannot be fully ruled out.

Evolutionary pattern of karyotypes and meiosis in pholcid spiders (Araneae: Pholcidae): implications for reconstructing chromosome evolution of araneomorph spiders.

BACKGROUND: Despite progress in genomic analysis of spiders, their chromosome evolution is not satisfactorily understood. Most information on spider chromosomes concerns the most diversified clade, entelegyne araneomorphs. Other clades are far less studied. Our study focused on haplogyne araneomorphs, which are remarkable for their unusual sex chromosome systems and for the co-evolution of sex chromosomes and nucleolus organizer regions (NORs); some haplogynes exhibit holokinetic chromosomes. To trace the karyotype evolution of haplogynes on the family level, we analysed the number and morphology of chromosomes, sex chromosomes, NORs, and meiosis in pholcids, which are among the most diverse haplogyne families. The evolution of spider NORs is largely unknown. RESULTS: Our study is based on an extensive set of species representing all major pholcid clades. Pholcids exhibit a low 2n and predominance of biarmed chromosomes, which are typical haplogyne features. Sex chromosomes and NOR patterns of pholcids are diversified. We revealed six sex chromosome systems in pholcids (X0, XY, X1X20, X1X2X30, X1X2Y, and X1X2X3X4Y). The number of NOR loci ranges from one to nine. In some clades, NORs are also found on sex chromosomes. CONCLUSIONS: The evolution of cytogenetic characters was largely derived from character mapping on a recently published molecular phylogeny of the family. Based on an extensive set of species and mapping of their characters, numerous conclusions regarding the karyotype evolution of pholcids and spiders can be drawn. Our results suggest frequent autosome-autosome and autosome-sex chromosome rearrangements during pholcid evolution. Such events have previously been attributed to the reproductive isolation of species. The peculiar X1X2Y system is probably ancestral for haplogynes. Chromosomes of the X1X2Y system differ considerably in their pattern of evolution. In some pholcid clades, the X1X2Y system has transformed into the X1X20 or XY systems, and subsequently into the X0 system. The X1X2X30 system of Smeringopus pallidus probably arose from the X1X20 system by an X chromosome fission. The X1X2X3X4Y system of Kambiwa probably evolved from the X1X2Y system by integration of a chromosome pair. Nucleolus organizer regions have frequently expanded on sex chromosomes, most probably by ectopic recombination. Our data suggest the involvement of sex chromosome-linked NORs in achiasmatic pairing.

Karyotype evolution and preliminary molecular assessment of genera in the family Scorpiopidae (Arachnida: Scorpiones).

The scorpions represent an ancient and morphologically conserved order of arachnids. Despite that, their karyotypes may differ considerably even among closely related species. In this study, we identify the trends of the karyotype evolution in the family Scorpiopidae based on integrating cytogenetic data and multi-locus molecular phylogenetic approaches. We detected considerable variability in diploid numbers of chromosomes (from 48 to 147), 18S rRNA gene cluster positions (from terminal to pericentromeric) at the interspecific level. Moreover, we identified independent fusions, fissions and inversions in the evolution of the family Scorpiopidae, leading to a remarkable diversification of the karyotypes. The dynamic system of the karyotype changes in this group is further documented by the presence of interstitial telomeric sequences (ITS) in two species. The cytogenetic differences observed among the analyzed species highlight the potential of this type of data for species-level taxonomy in scorpion lineages with monocentric chromosomes. Additionally, the results of our phylogenetic analyses support the monophyly of the family Scorpiopidae, but rendered several genera para- or polyphyletic.

Cytogenetics of entelegyne spiders (Arachnida, Araneae) from southern Africa.

Spiders represent one of the most studied arachnid orders. They are particularly intriguing from a cytogenetic point of view, due to their complex and dynamic sex chromosome determination systems. Despite intensive research on this group, cytogenetic data from African spiders are still mostly lacking. In this study, we describe the karyotypes of 38 species of spiders belonging to 16 entelegyne families from South Africa and Namibia. In the majority of analysed families, the observed chromosome numbers and morphology (mainly acrocentric) did not deviate from the family-level cytogenetic characteristics based on material from other continents: Tetragnathidae (2n♂ = 24), Ctenidae and Oxyopidae (2n♂ = 28), Sparassidae (2n♂ = 42), Gnaphosidae, Trachelidae and Trochanteriidae (2n♂ = 22), and Salticidae (2n♂ = 28). On the other hand, we identified interspecific variability within Hersiliidae (2n♂ = 33 and 35), Oecobiidae (2n♂ = 19 and 25), Selenopidae (2n♂ = 26 and 29) and Theridiidae (2n♂ = 21 and 22). We examined the karyotypes of Ammoxenidae and Gallieniellidae for the first time. Their diploid counts (2n♂ = 22) correspond to the superfamily Gnaphosoidea and support their placement in this lineage. On the other hand, the karyotypes of Prodidominae (2n♂ = 28 and 29) contrast with all other Gnaphosoidea. Similarly, the unusually high diploid number in Borboropactus sp. (2n♂ = 28) within the otherwise cytogenetically uniform family Thomisidae (mainly 2n♂ = 21-24) supports molecular data suggesting a basal position of the genus in the family. The implementation of FISH methods for visualisation of rDNA clusters facilitated the detection of complex dynamics of numbers of these loci. We identified up to five loci of the 18S rDNA clusters in our samples. Three different sex chromosome systems (X0, X1X20 and X1X2X30) were also detected among the studied taxa.

Cryptic diversity and dynamic chromosome evolution in Alpine scorpions (Euscorpiidae: Euscorpius).

Over time, mountain biota has undergone complex evolutionary histories that have left imprints on its genomic arrangement, geographical distribution and diversity of contemporary lineages. Knowledge on these biogeographical aspects still lags behind for invertebrates inhabiting the Alpine region. In the present study, we examined three scorpion species of the subgenus Euscorpius (Alpiscorpius) from the European Alps using cytogenetic and molecular phylogenetic approaches to determine the variation and population structure of extant lineages at both chromosome and genetic level, and to provide an insight into the species diversification histories. We detected considerable intraspecific variability in chromosome complements and localization of the 18S rDNA loci in all studied species. Such chromosome differences were noticeable as the existence of three [in E. (A.) alpha and E. (A.) germanus] or four [in E. (A.) gamma] range-restricted karyotypic races. These races differed from one another either by 2n [in E. (A.) alpha 2n = 54, 60, 90; in E. (A.) gamma 2n = 58, 60, 88, 86-92], or by the karyotypic formula [in E. (A.) germanus 2n = 34m + 12sm; 36m + 10sm; 42m + 4sm]. Using mitochondrial (16S rRNA, COI) and nuclear (28S rDNA) genetic markers, we examined genetic variation and reconstructed phylogenetic relationships among the karyotypic races. Both approaches provided evidence for the existence of ten deeply divergent lineages exhibiting the features of local endemics and indicating the presence of cryptic species. Molecular dating analyses suggest that these lineages diversified during the Plio-Pleistocene and this process was presumably accompanied by dynamic structural changes in the genome organization.

Morphological and cytogenetic characteristics of Neobisium (Blothrus) slovacum Gulicka, 1977 (Pseudoscorpiones, Neobisiidae), the northernmost troglobitic species of the subgenus Blothrus in Europe.

A redescription is provided of the adult, tritonymph and deutonymph life stages of the troglobitic Neobisium (Blothrus) slovacum Gulicka, 1977, which is known from Slovakia and Hungary. Material examined included 35 previously deposited museum specimens and 15 newly collected specimens. In addition, the karyotype and distribution of 18S rDNA clusters are described, using fluorescence in situ hybridization (FISH). The male karyotype of N.slovacum comprises 69 chromosomes, with a predominance of biarmed chromosomes, and an X0 sex chromosome system. Two pairs of signals for 18S rDNA on biarmed chromosomes (submetacentric and metacentric) of different sizes were identified. The present study provides the first information about the distribution of these clusters in the arachnid order Pseudoscorpiones. The geographic distribution of the species is summarized and mapped. Neobisiumslovacum is endemic to the Slovak and Aggtelek Karst area in southern Slovakia and north-western Hungary, where it has been recorded from 16 caves. One of these, Hacavská cave (in Slovakia), is the northernmost locality known for any species of the subgenus Blothrus.

Molecular technique reveals high variability of 18S rDNA distribution in harvestmen (Opiliones, Phalangiidae) from South Africa.

The knowledge of cytogenetics in the harvestmen family Phalangiidae has been based on taxa from the Northern Hemisphere. We performed cytogenetic analysis on Guruia africana (Karsch, 1878) (2n=24) and four species of the genus Rhampsinitus Simon, 1879 (2n=24, 26, 34) from South Africa. Fluorescence in situ hybridization with an 18S rDNA probe was used to analyze the number and the distribution of this cluster in the family Phalangiidae for the first time. The results support the cytogenetic characteristics typical for the majority of harvestmen taxa, i.e. the predominance of small biarmed chromosomes and the absence of morphologically well-differentiated sex chromosomes as an ancestral state. We identified the number of 18S rDNA sites ranging from two in R. qachasneki Kauri, 1962 to seven in one population of R. leighi Pocock, 1903. Moreover, we found differences in the number and localization of 18S rDNA sites in R. leighi between populations from two localities and between sexes of R. capensis (Loman, 1898). The heterozygous states of the 18S rDNA sites in these species may indicate the presence of XX/XY and ZZ/ZW sex chromosomes, and the possible existence of these systems in harvestmen is discussed. The variability of the 18S rDNA sites indicates intensive chromosomal changes during the differentiation of the karyotypes, which is in contrast to the usual uniformity in chromosomal morphology known from harvestmen so far.

Karyotype diversity of pseudoscorpions of the genus Chthonius (Pseudoscorpiones, Chthoniidae) in the Alps.

Pseudoscorpions are found in almost all terrestrial habitats. However, their uniform appearance presents a challenge for morphology-based taxonomy, which may underestimate the diversity of this order. We performed cytogenetic analyses on 11 pseudoscorpion species of the genus Chthonius C. L. Koch, 1843 from the Alps, including three subgenera: Chthonius (Chthonius) C. L. Koch, 1843, Chthonius (Ephippiochthonius) Beier, 1930 and Chthonius (Globochthonius) Beier, 1931 inhabiting this region. The results show that the male diploid number of chromosomes ranges from 21-35. The sex chromosome system X0 has been detected in all male specimens. The X sex chromosome is always metacentric and represents the largest chromosome in the nucleus. Achiasmatic meiosis, already known from the family Chthoniidae, was further confirmed in males of Chthonius. C-banding corroborated the localization of constitutive heterochromatin in the centromere region, which corresponds to heteropycnotic knobs on the standard chromosome preparations. Morphological types and size differentiation of chromosomes in the karyotype suggest that the main chromosomal rearrangements in the evolution of Chthonius are centric or tandem fusions resulting in a decrease in the number of chromosomes. Pericentric inversions, inducing the change of acrocentric chromosomes into biarmed chromosomes, could also be expected. Variability in chromosome morphology and number was detected in several species: Chthonius (Chthonius) ischnocheles (Hermann, 1804), Chthonius (Chthonius) raridentatus, Chthonius (Chthonius) rhodochelatus Hadzi, 1930, and Chthonius (Chthonius) tenuis L. Koch, 1873. We discuss the intraspecific variability within these species and the potential existence of cryptic species.

Karyotype Evolution in Harvestmen of the Suborder Cyphophthalmi (Opiliones).

The morphologically uniform suborder Cyphophthalmi represents a basal group of harvestmen (Opiliones). As such, it plays an important role in the reconstruction of the karyotype evolution within this arachnid order. The cytogenetic analysis of 6 representatives of the suborder Cyphophthalmi, namely Miopsalis sp. (2n = 30; Stylocellidae), Austropurcellia arcticosa (Cantrell, 1980) (2n = 30; Pettalidae), Parapurcellia amatola de Bivort & Giribet, 2010 (2n = 32; Pettalidae), Paramiopsalis aff. ramulosus Juberthie, 1962 (2n = 28; Sironidae), Cyphophthalmus duricorius Joseph, 1868 (2n = 24; Sironidae), and Siro carpaticus Rafalski, 1956 (2n = 52; Sironidae) was performed. Fluorescence in situ hybridization with 18S rDNA probe was used to analyze the distribution of major ribosomal RNA genes in harvestmen. We confront the obtained cytogenetic data with current hypotheses on cyphophthalmid phylogeny to reconstruct their karyotype evolution. We conclude that the ancestral karyotype of harvestmen consisted of 2n = 30 elements with 1 chromosome pair bearing terminal rDNA clusters. The rDNA locus was multiplicated in the evolution of Cyphophthalmi. However, decreases as well as increases in the number of chromosomes have been detected in the karyotype evolution of Cyphophthalmi. Our data thus reveal unexpected diversity in cyphophthalmid karyotypes.

Comparison of different cytogenetic methods and tissue suitability for the study of chromosomes in Cimex lectularius (Heteroptera, Cimicidae).

In the article we summarize the most common recent cytogenetic methods used in analysis of karyotypes in Heteroptera. We seek to show the pros and cons of the spreading method compared with the traditional squashing method. We discuss the suitability of gonad, midgut and embryo tissue in Cimex lectularius Linnaeus, 1758 chromosome research and production of figures of whole mitosis and meiosis, using the spreading method. The hotplate spreading technique has many advantages in comparison with the squashing technique. Chromosomal slides prepared from the testes tissue gave the best results, tissues of eggs and midgut epithelium are not suitable. Metaphase II is the only division phase in which sex chromosomes can be clearly distinguished. Chromosome number determination is easy during metaphase I and metaphase II. Spreading of gonad tissue is a suitable method for the cytogenetic analysis of holokinetic chromosomes of Cimex lectularius.

Extensive fragmentation of the X chromosome in the bed bug Cimex lectularius Linnaeus, 1758 (Heteroptera, Cimicidae): a survey across Europe.

Variation in the number of chromosomes was revealed in 61 samples of Cimex lectularius Linnaeus, 1758 from the Czech Republic and other European countries, hosted on Myotis Kaup, 1829 (4) and Homo sapiens Linnaeus, 1758 (57). The karyotype of all the specimens of Cimex lectularius analysed contained 26 autosomes and a varying number of the sex chromosomes. The number of sex chromosomes showed extensive variation, and up to 20 fragments were recorded. Altogether, 12 distinct karyotypes were distinguished. The male karyotypes consisted of 29, 30, 31, 32, 33, 34, 35, 36, 37, 40, 42 and 47 chromosomes. The females usually exhibited the number of chromosomes which was complementary to the number established in the males from the same sample. However, 11 polymorphic samples were revealed in which the karyotypes of females and males were not complementary each other. The complement with 2n = 26+X1X2Y was found in 44% of the specimens and 57,4% samples of bed bugs studied. The karyotypes with higher chromosome numbers as well as individuals with chromosomal mosaics were usually found within the samples exhibiting particularly extensive variation between individuals, and such complements were not found within samples contaning a few or single specimen. The occurrence of chromosomal mosaics with the karyotype constitution varying between cells of single individual was observed in five specimens (4.3%) from five samples. We assume that polymorphism caused by fragmentation of the X chromosome may result in meiotic problems and non-disjunction can produce unbalanced gametes and result in lowered fitness of individuals carrying higher numbers of the X chromosome fragments. This effect should be apparently enhanced with the increasing number of the fragments and this may be the reason for the observed distribution pattern of individual karyotypes in the studied samples and the rarity of individuals with extremely high chromosome numbers. The assumed lowering of the fitness of individuals carrying higher numbers of the X chromosome fragments could affect population dynamics of variable populations.

Karyotype study on pseudoscorpions of the genus Lasiochernes Beier (Pseudoscorpiones, Chernetidae).

Karyotypes of the genus Lasiochernes (Pseudoscorpiones, Chernetidae) are studied for the first time. The diploid chromosome numbers of males were found to be 2n = 61 in L. pilosus, 2n = 69 in L. siculus and 2n = 73 in L. cretonatus. Karyotypes of all species mainly consist of biarmed chromosomes; the sex chromosome system is XO. Remarkably, the X chromosome displays partial (L. cretonatus), or even total (L. pilosus), negative heteropycnosis during the spermatogonial metaphase.

Karyotype analysis and achiasmatic meiosis in pseudoscorpions of the family Chthoniidae (Arachnida: Pseudoscorpiones).

Karyotypes of pseudoscorpions (Arachnida, Pseudoscorpiones) are largely unknown. Here we describe for the first time karyotypes of the suborder Epiocheirata, represented by 9 European species of two genera of Chthoniidae, Chthonius and Mundochthonius. Diploid chromosome numbers of males range from 21 to 37. Karyotypes of both genera differ substantially. Acrocentric chromosomes predominate in karyotypes of the genus Chthonius, whereas M. styriacus exhibits a predominance of metacentric chromosomes. These differences suggest that the two genera belong probably to distant branches of the family Chthoniidae. It is proposed that karyotype evolution of the genus Chthonius was characterised by a reduction of chromosome numbers by tandem and centric fusions as well as gradual conversion of acrocentric chromosomes to biarmed ones, mostly by pericentric inversions. A tendency towards reduced chromosome numbers is evident in the subgenus Ephippiochthonius. All species display X0 sex chromosome system that is probably ancestral in pseudoscorpions. The X chromosome exhibits conservative morphology. It is metacentric in all species examined, and in the majority of them, a subterminal secondary constriction was found at one of its arms. In contrast to chthoniids, secondary constriction was not reported on sex chromosomes of other pseudoscorpions. Analysis of prophase I chromosomes in males revealed an achiasmatic mode of meiosis. Findings of the achiasmatic meiosis in both genera, Chthonius and Mundochthonius, indicate that this mode of meiosis might be characteristic of the family Chthoniidae. Amongst arachnids, achiasmatic meiosis has only been described in some scorpions, acariform mites, and spiders.