Marielle Cristina Schneider

Complex meiotic configuration of the holocentric chromosomes: the intriguing case of the scorpion Tityus bahiensis

Mitotic and meiotic chromosomes of Tityus bahiensis were investigated using light (LM) and transmission electron microscopy (TEM) to determine the chromosomal characteristics and disclose the mechanisms responsible for intraspecific variability in chromosome number and for the presence of complex chromosome association during meiosis. This species is endemic to Brazilian fauna and belongs to the family Buthidae, which is considered phylogenetically basal within the order Scorpiones. In the sample examined, four sympatric and distinct diploid numbers were observed: 2n = 5, 2n = 6, 2n = 9, and 2 = 10. The origin of this remarkable chromosome variability was attributed to chromosome fissions and/or fusions, considering that the decrease in chromosome number was concomitant with the increase in chromosome size and vice versa. The LM and TEM analyses showed the presence of chromosomes without localised centromere, the lack of chiasmata and recombination nodules in male meiosis, and two nucleolar organiser regions carrier chromosomes. Furthermore, male prophase I cells revealed multivalent chromosome associations and/or unsynapsed or distinctly associated chromosome regions (gaps, less-condensed chromatin, or loop-like structure) that were continuous with synapsed chromosome segments. All these data permitted us to suggest that the chromosomal rearrangements of T. bahiensis occurred in a heterozygous state. A combination of various factors, such as correct disjunction and balanced segregation of the chromosomes involved in complex meiotic pairing, system of achiasmate meiosis, holocentric nature of the chromosomes, population structure, and species dispersion patterns, could have contributed to the high level of chromosome rearrangements present in T. bahiensis.

High chromosome variability and the presence of multivalent associations in buthid scorpions

In this study, we investigated the mitotic and meiotic chromosomes of 11 Buthidae scorpion species, belonging to three genera (Ananteris, Rhopalurus and Tityus), to obtain detailed knowledge regarding the mechanisms underlying the intraspecific and/or interspecific diversity of chromosome number and the origin of the complex chromosome associations observed during meiosis. The chromosomes of all species did not exhibit a localised centromere region and presented synaptic and achiasmatic behaviour during meiosis I. Spermatogonial and/or oogonial metaphase cells of these buthids showed diploid numbers range from 2n = 6 to 2n = 28. In most species, multivalent chromosome associations were observed in pachytene and postpachytene nuclei. Moreover, intraspecific variability associated with the presence or absence of chromosome chains and the number of chromosomes in the complex meiotic configurations was observed in some species of these three genera. Silver-impregnated cells revealed that the number and location of nucleolar organiser regions (NORs) remained unchanged despite extensive chromosome variation; notably, two NORs located on the terminal or subterminal chromosome regions were commonly observed for all species. C-banded and fluorochrome-stained cells showed that species with conspicuous blocks of heterochromatin exhibited the lowest rate of chromosomal rearrangement. Based on the investigation of mitotic and meiotic cells, we determined that the intraspecific variability occurred as a consequence of fission/fusion-type chromosomal rearrangements in Ananteris and Tityus species and reciprocal translocation in Rhopalurus species. Furthermore, we verified that individuals presenting the same diploid number differ in structural chromosome organisation, giving rise to intraspecific differences of chromosome association in meiotic cells (bivalent-like elements or chromosome chains).

Chromosomes of Theridiidae spiders (Entelegynae): interspecific karyotype diversity in Argyrodes and diploid number intraspecific variability in Nesticodes rufipes

Theridiidae is a derived family within the Araneoidea clade. In contrast to closely related groups, the 2n(male) = 20+X1 X 2 with acro/telocentric chromosomes is the most widespread karyotype among the theridiid spiders. In this work, the cytogenetic analysis of Argyrodes elevatus revealed original chromosome features different from those previously registered for Theridiidae, including the presence of 2n(male) = 20+X with meta/submetacentric chromosomes. Most individuals of Nesticodes rufipes showed family conserved karyotype characteristics. However, one individual had a 2n(male) = 24 due to the presence of an extra chromosome pair, which exhibited regular behavior and reductional segregation during meiosis. After silver staining, mitotic cells exhibited NORs localized on the terminal regions of the short arms of pairs 2, 3, and 4 of A. elevatus and on the terminal regions of long arms of pair 4 of N. rufipes. The comparative analysis with data from phylogenetically related species allowed the clarification of the origin of the interspecific and intraspecific chromosome variability observed in Argyrodes and in N. rufipes, respectively.

Karyotype plasticity in crickets: Numerical, morphological, and nucleolar organizer region distribution pattern of Anurogryllus sp.

Abstract Within the Orthopteran species, those of the suborder Ensifera have been rarely studied from the cytogenetic point of view, mainly due to the difficulties for taxonomic identification of its species. The Gryllidae is the second largest family of this suborder and possesses some genera, such as Anurogryllus, that occur only on the American continents. The aim of this work was to determine the karyotype characteristics, the meiotic chromosome behaviour, and the nucleolar organizer region (NOR) pattern of Anurogryllus sp (Orthoptera: Gryllidae). In the analyzed sample, high levels of numerical, morphological, and NORs polymorphisms were detected. Within five distinct karyotypes that were found, the basic karyotype of Anurogryllus sp. showed 2n(♂) = 22 + X0 with acrocentric autosomes and a metacentric X sex chromosome; furthermore, a conspicuous secondary constriction related to the NOR was present along the entire short arm on pair 5. The other four types of karyotypes arose from centric fusions between elements of pairs 1/3, 2/6, 4/7 and a NOR partial translocation from pair 5 onto the long arm terminal region of one element of the fused pair 2/6. Such intraspecific variability and the consequences of high levels of polymorphism are discussed, leading to conjectures about the mechanisms that led to these chromosome rearrangements.

Chromosomal similarities between Nephilidae and Tetragnathidae indicate unique evolutionary traits among Araneoidea

Abstract Nephilid systematics has been subject to several changes in the last years, and the use of non-classical characters could be useful for evolutionary considerations. In this study, we analyzed the mitotic chromosomes of two nephilid spiders, Nephila clavipes and Nephila sexpunctata, using standard staining, silver nitrate impregnation and C-banding techniques, aiming to discuss the chromosomal similarities of Nephilidae and Tetragnathidae, and chromosome evolution within Nephila and Nephilingis. The basic karyotype characteristics observed in these two species (2n♂ = 22 + X1X20 and monoarmed chromosomes) were similar to those registered for most araneoid families, i.e., Araneidae, Linyphiidae, Nephilidae, Nesticidae and Tetragnathidae. However, the occurrence of both prominent secondary constrictions and nucleolar organizer regions (NORs) is a shared characteristic between Nephilidae and Tetragnathidae, considering that these regions were not observed in any other Araneoidea species cytogenetically examined. Furthermore, in the present study we showed that within Nephila and Nephilingis species, change in the number and location of NORs as well as in the quantity and distribution of constitutive heterochromatin were the main events responsible for chromosome evolution, and that these differences can be useful in the cytotaxonomy of this group.

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

Abstract Even though less than 2% of pholcid species have been karyotyped, previous studies documented a wide diversity of diploid numbers and sex chromosome systems. Here, we increase the number of native Brazilian cytogenetically investigated pholcid species from three to eight and discuss implications of chromosome evolution in this group. The species analyzed here share a X0/XX sex chromosome system and biarmed chromosomes, but vary in diploid numbers, i.e., 2n♂ = 17 in Mesabolivar spinulosus (Mello-Leitão, 1939) and Mesabolivar togatus (Keyserling, 1891), 2n♂ = 15 in Carapoia sp., and 2n♂ = 9 in Micropholcus piaui Huber, Carvalho & Benjamin, 2014 and Micropholcus ubajara Huber, Carvalho & Benjamin, 2014. Chromosomal data indicate that most Mesabolivar species share a 2n♂ = 17, X0, while Mesabolivar luteus shares with Carapoia sp. a 2n♂ = 15, X0. This lends further support to the idea that M. luteus is in fact misplaced and more closely related to Carapoia González-Sponga, 1998. The diploid number of the two Micropholcus species is the lowest reported so far for spiders with monocentric chromosomes. The 2n♂ = 9, differs strongly from the 2n♂ = 17 previously reported for Micropholcus fauroti (Simon, 1887). As the number of autosomes of M. piaui and M. ubajara is exactly half of that found in M. fauroti, we hypothesize that the reduction occurred by an “all or nothing” fusion event. The low diploid number observed in M. piaui and M. ubajara is the first morphological synapomorphy that would support the establishment of a new genus to allocate the New World Micropholcus species.

Mechanisms of karyotype evolution in the Brazilian scorpions of the subfamily Centruroidinae (Buthidae)

The recently-revised subfamily Centruroidinae is part of the New World clade of buthid scorpions. In this study, we analyzed the cytogenetic characteristics of nine of the 10 Brazilian centruroidines, and one undescribed species of the genus Ischnotelson, using a phylogenetic approach to determine the chromosomal rearrangements responsible for the differentiation of karyotypes among the species. The cytogenetic data recorded in the present study supported the new taxonomic arrangement of the Centruroidinae, with all the species of the same genus sharing the same or similar diploid numbers, i.e., 2n = 20 or 22 in Troglorhopalurus lacrau and T. translucidus, 2n = 25 or 26 in Ischnotelson sp., I. guanambiensis and I. peruassu, and 2n = 28 in Jaguajir agamemnon, J. pintoi and J. rochae. The karyotype modelling in the ChromEvol software indicated 2n = 18 as the ancestral diploid number of the Centruroidinae. The differentiation of karyotypes among the centruroidine genera was based on increasing chromosome numbers resulting from progressive fission events. These changes probably occurred prior to the diversification of the genera Ischnotelson, Jaguajir, Physoctonus and Rhopalurus, and appear to have played a more important role in karyotype evolution at the intergeneric level than the interspecific one. However, the observed increase in diploid numbers was not accompanied by changes in the number or location of ribosomal genes or telomeric sequences. The identification of meiotic cells in female specimens also allowed us to discuss the mechanisms of achiasmatic meiosis in scorpions.

Presence and location of TTAGG telomeric sequence in holocentric chromosomes of buthid scorpions

Universidade Estadual Paulista Julio de Mesquita Filho, Instituto de Biociencias, Rio Claro, BrasilThe advent of the next generation sequencing (NGS) made sequencing and scaffolding of an entire animal genome a routine procedure. As the result we face a fast increase in the number of animal genomes available due to the activities of large international genome sequencing initiatives e.g., Genome 10K (G10K) or smaller projects. However, the full informative power of a sequenced genome could only be achieved when it is assembled into chromosomes. Usually, a draft or nearly complete animal chromosome assembly is achieved through three steps: (i) constructing contigs based on read overlaps, (ii) merging contigs into scaffolds using pair-end reads, and (iii) mapping scaffolds on chromosomes with the use of physical or genetic maps. As the cost of mapping techniques is still much higher than sequencing, the genetic and physical maps are not available for the majority of the de novo sequenced genomes. To overcome this problem for assemblies that employ long-insert libraries (5 – 40 Kbp) we recently developed the reference-assisted chromosome assembly (RACA) algorithm (Kim et al., 2013). This method relies on both the raw sequencing data (reads) and comparative information; the latter is obtained from alignments between the target (de novo sequenced), a closely related (reference) and more distantly related (outgroup) genomes. Using RACA followed by the manual FISH or PCR verification steps we are reconstructing the chromosome organisation of 19 bird species sequenced by the G10K community. We use the publically available chicken (Gallus gallus) and zebra finch (Taeniopygia guttata) chromosome assemblies as either reference or outgroup for each reconstruction depending on their phylogenetic relationships with each target species. Initially, we established the optimal RACA parameters for a bird chromosome assembly reconstruction using the duck (Anas platyrhynchos) and budgerigar (Melopsittacus undulatus) super-scaffolds assembled with the support from physical maps. This step allowed us to test the reliability of RACA reconstructions for bird genomes. Due to a higher evolutionary conservation of the bird karyotype compared to the mammalian one, we have achieved ~97% accuracy of scaffold adjacencies in our predicted chromosome fragments compared to the ~93-96% accuracies reported for mammals (Kim et al., 2013). We detected ~4-28% of scaffolds in different target bird genomes that are either chimeric or containing genuine lineage-specific evolutionary breakpoint regions. Some of these scaffolds will be selected for follow up PCR or FISH verifications. All RACA reconstructions will become publicly available from our Evolution Highway comparative chromosome browser http://evolutionhighway.ncsa.uiuc.edu/birds/ and will be further utilised to study connections between the chromosome evolution, adaptation and phenotypic diversity in birds and other vertebrates.Universidade Estadual Paulista, Nucleo de Pesquisa e Conservacao de Cervideos, Faculdade de Ciencias Agrarias e Veterinarias, Jaboticabal, Brasil• Invited speaker abstracts have the prefix “S” • Selected Oral presentations have the prefix “O” • Poster abstracts have the prefix “P” S1: 50th Anniversary of the first Oxford Chromosome Conference and some reflections on chromosome synapsis Malcolm A Ferguson-Smith Department of Veterinary Medicine, University of Cambridge, Cambridge, UK. Cyril Darlington, who organised the first Oxford Chromosome Conference 50 years ago, was one of the great pioneers of cytogenetics. He brought new understanding to the mechanisms of mitosis and meiosis and the uniformity of chromosome behaviour in all plants and animals with its implications for evolution. His major conclusions relate to the origin of chiasmata and the properties of sex chromosomes and were based on light microscopy before the era of molecular cytogenetics in the 1970s. Since his day the field has been transformed by electron microscopy, FISH, immunofluorescence of chromosomal proteins, meiotic mutants in yeast and mice and by DNA mapping and sequencing. Progress in understanding chromosome structure and synapsis in meiotic and somatic cells since Darlington will be briefly summarised with emphasis on the unknown. Genome conservation and the nature of non-coding DNA at synapsis sites, and the threedimensional structure of chromosomal proteins (eg, those of axial filaments), should be among the topics for discussion at this and future Chromosome Conferences. 20th International Chromosome Conference (ICCXX) 35120th International Chromosome Conference (ICCXX). 50th Anniversary, University of Kent, Canterbury, 1st–4th September 2014.Dinosaurs hold a unique place both in the history of the earth and the imagination of many. They dominated the terrestrial environment for around 170 million years during which time they diversified into at least 1000 different species. Reptilia, within which they are placed is one of the most remarkable vertebrate groups, consisting of two structurally and physiologically distinct lineages – the birds and the non-avian reptiles, of which there are 10,000 and 7,500 extant species respectively. The dinosaurs are without doubt the most successful group of vertebrate to have existed. They survived several mass extinction events before finally non-avian dinosaurs were defeated 66 million years ago in the Cretaceous-Paleogene extinction event, leaving the neornithes (modern birds) as their living descendants. Aside from the huge phenotypic diversity seen in this group, the birds and non-avian reptiles interestingly display similar karyotypic patterns (with the exception of crocodilians); with the characteristic pattern of macro and micro chromosomes, small genome size and few repetitive elements, suggesting that these were features exhibited in their common ancestor. In this study, the availability of multiple reptile genome sequences (including birds) on an interactive browser (Evolution Highway) allowed us to identify multi species homologous synteny blocks (msHSBs) between the putative avian ancestor (derived from six species of extant birds), the Lizard (Anolis carolensis) and the Snake (Boa constrictor). From these msHSBs we were able to produce a series of contiguous ancestral regions (CARs) representing the most likely ancestral karyotype of the Saurian (ancestor of archosaurs and lepidosaurs) that diverged from the mammalian lineage 280 mya. From this we have hypothesised the series of inter and intra-chromosomal rearrangements that have occurred along the dinosaur (archosaur) lineage to the ancestor of modern birds (100 mya) and along the lepidosaur lineage to the modern snake and lizard using the model of maximum parsimony. Our study shows that relatively few chromosomal rearrangements took place over this period with an average of one inter or intra-chromosomal (translocations and inversions respectively) rearrangement occurring approximately every 2 million years. The majority of these rearrangements appear to be intra-chromosomal suggesting an overall karyotypic stability, which is consistent with that of that of modern birds. Our results support the hypothesis that the characteristically avian genome was present in the saurian ancestor and that it has remained remarkably stable in the 280 million years since. It is credible therefore to suggest that this ‘avian-style’ genome may be one of the key factors in the success of this extraordinarily diverse animal group.

Mitotic and meiotic chromosomes of Tityus mattogrossensis and Tityus silvestris (Scorpiones, Buthidae)

Universidade Estadual Paulista Julio de Mesquita Filho, Instituto de Biociencias, Rio Claro, BrasilThe advent of the next generation sequencing (NGS) made sequencing and scaffolding of an entire animal genome a routine procedure. As the result we face a fast increase in the number of animal genomes available due to the activities of large international genome sequencing initiatives e.g., Genome 10K (G10K) or smaller projects. However, the full informative power of a sequenced genome could only be achieved when it is assembled into chromosomes. Usually, a draft or nearly complete animal chromosome assembly is achieved through three steps: (i) constructing contigs based on read overlaps, (ii) merging contigs into scaffolds using pair-end reads, and (iii) mapping scaffolds on chromosomes with the use of physical or genetic maps. As the cost of mapping techniques is still much higher than sequencing, the genetic and physical maps are not available for the majority of the de novo sequenced genomes. To overcome this problem for assemblies that employ long-insert libraries (5 – 40 Kbp) we recently developed the reference-assisted chromosome assembly (RACA) algorithm (Kim et al., 2013). This method relies on both the raw sequencing data (reads) and comparative information; the latter is obtained from alignments between the target (de novo sequenced), a closely related (reference) and more distantly related (outgroup) genomes. Using RACA followed by the manual FISH or PCR verification steps we are reconstructing the chromosome organisation of 19 bird species sequenced by the G10K community. We use the publically available chicken (Gallus gallus) and zebra finch (Taeniopygia guttata) chromosome assemblies as either reference or outgroup for each reconstruction depending on their phylogenetic relationships with each target species. Initially, we established the optimal RACA parameters for a bird chromosome assembly reconstruction using the duck (Anas platyrhynchos) and budgerigar (Melopsittacus undulatus) super-scaffolds assembled with the support from physical maps. This step allowed us to test the reliability of RACA reconstructions for bird genomes. Due to a higher evolutionary conservation of the bird karyotype compared to the mammalian one, we have achieved ~97% accuracy of scaffold adjacencies in our predicted chromosome fragments compared to the ~93-96% accuracies reported for mammals (Kim et al., 2013). We detected ~4-28% of scaffolds in different target bird genomes that are either chimeric or containing genuine lineage-specific evolutionary breakpoint regions. Some of these scaffolds will be selected for follow up PCR or FISH verifications. All RACA reconstructions will become publicly available from our Evolution Highway comparative chromosome browser http://evolutionhighway.ncsa.uiuc.edu/birds/ and will be further utilised to study connections between the chromosome evolution, adaptation and phenotypic diversity in birds and other vertebrates.Universidade Estadual Paulista, Nucleo de Pesquisa e Conservacao de Cervideos, Faculdade de Ciencias Agrarias e Veterinarias, Jaboticabal, Brasil• Invited speaker abstracts have the prefix “S” • Selected Oral presentations have the prefix “O” • Poster abstracts have the prefix “P” S1: 50th Anniversary of the first Oxford Chromosome Conference and some reflections on chromosome synapsis Malcolm A Ferguson-Smith Department of Veterinary Medicine, University of Cambridge, Cambridge, UK. Cyril Darlington, who organised the first Oxford Chromosome Conference 50 years ago, was one of the great pioneers of cytogenetics. He brought new understanding to the mechanisms of mitosis and meiosis and the uniformity of chromosome behaviour in all plants and animals with its implications for evolution. His major conclusions relate to the origin of chiasmata and the properties of sex chromosomes and were based on light microscopy before the era of molecular cytogenetics in the 1970s. Since his day the field has been transformed by electron microscopy, FISH, immunofluorescence of chromosomal proteins, meiotic mutants in yeast and mice and by DNA mapping and sequencing. Progress in understanding chromosome structure and synapsis in meiotic and somatic cells since Darlington will be briefly summarised with emphasis on the unknown. Genome conservation and the nature of non-coding DNA at synapsis sites, and the threedimensional structure of chromosomal proteins (eg, those of axial filaments), should be among the topics for discussion at this and future Chromosome Conferences. 20th International Chromosome Conference (ICCXX) 35120th International Chromosome Conference (ICCXX). 50th Anniversary, University of Kent, Canterbury, 1st–4th September 2014.Dinosaurs hold a unique place both in the history of the earth and the imagination of many. They dominated the terrestrial environment for around 170 million years during which time they diversified into at least 1000 different species. Reptilia, within which they are placed is one of the most remarkable vertebrate groups, consisting of two structurally and physiologically distinct lineages – the birds and the non-avian reptiles, of which there are 10,000 and 7,500 extant species respectively. The dinosaurs are without doubt the most successful group of vertebrate to have existed. They survived several mass extinction events before finally non-avian dinosaurs were defeated 66 million years ago in the Cretaceous-Paleogene extinction event, leaving the neornithes (modern birds) as their living descendants. Aside from the huge phenotypic diversity seen in this group, the birds and non-avian reptiles interestingly display similar karyotypic patterns (with the exception of crocodilians); with the characteristic pattern of macro and micro chromosomes, small genome size and few repetitive elements, suggesting that these were features exhibited in their common ancestor. In this study, the availability of multiple reptile genome sequences (including birds) on an interactive browser (Evolution Highway) allowed us to identify multi species homologous synteny blocks (msHSBs) between the putative avian ancestor (derived from six species of extant birds), the Lizard (Anolis carolensis) and the Snake (Boa constrictor). From these msHSBs we were able to produce a series of contiguous ancestral regions (CARs) representing the most likely ancestral karyotype of the Saurian (ancestor of archosaurs and lepidosaurs) that diverged from the mammalian lineage 280 mya. From this we have hypothesised the series of inter and intra-chromosomal rearrangements that have occurred along the dinosaur (archosaur) lineage to the ancestor of modern birds (100 mya) and along the lepidosaur lineage to the modern snake and lizard using the model of maximum parsimony. Our study shows that relatively few chromosomal rearrangements took place over this period with an average of one inter or intra-chromosomal (translocations and inversions respectively) rearrangement occurring approximately every 2 million years. The majority of these rearrangements appear to be intra-chromosomal suggesting an overall karyotypic stability, which is consistent with that of that of modern birds. Our results support the hypothesis that the characteristically avian genome was present in the saurian ancestor and that it has remained remarkably stable in the 280 million years since. It is credible therefore to suggest that this ‘avian-style’ genome may be one of the key factors in the success of this extraordinarily diverse animal group.