Backcross Males Research Articles

Enhancing aggression in Henan gamecocks via augmentation of serotonergic-dopaminergic signaling and attenuation of neuroimmune response

Animal aggression is one of the most conserved behaviors. Excessive and inappropriate aggression was a serious social concern across species. After long-term selection under strict stress conditions, Henan gamecock serves as a good model for studying aggressive behavior. In this research, we constructed a Henan game chicken backcross population containing 25% Rhode Island Red (RIR), and conducted brain transcriptomics and serum metabolomics analyses on Henan gamecock (HGR) through its comparison with its female encounters (HGH) and the male backcross birds (BGR). The study revealed that seven differential metabolites in serum and 172 differentially expressed genes in the brain were commonly shared in both HGR vs. HGH and HGR vs. BGR comparisons. They exhibited the same patterns of modulation in Henan gamecocks, following either HGH < HGR > BGR or HGH > HGR < BGR style. Therein, some neurological genes involving in serotonergic and dopaminergic signaling were upregulated, while the levels of many genes related with neuro-immune function were decreased in Henan gamecock. In addition, many unknown genes specifically or highly expressed in the brain of the Henan gamecock were identified. These genes are potentially key candidates for enhancing the bird's aggression. Multi-omics joint analysis revealed that tyrosine metabolism and neuroactive ligand-receptor interaction were commonly affected. Overall, our results propose that the aggressiveness of Henan gamecocks can be heightened by the activation of the serotonergic-dopaminergic metabolic process in the brain, which concurrently impairs the neuroimmune system. Further research is needed to identify the function of these unknown genes on the bird's aggressive behavior.

Evaluating evidence of mitonuclear incompatibilities with the sex chromosomes in an avian hybrid zone.

The exploration of hybrid zones and the intergenomic conflicts exposed through hybridization provide windows into the processes of divergence and speciation. Sex chromosomes and mitonuclear incompatibilities have strong associations with the genetics of hybrid dysfunction. In ZW sex-determining systems, maternal co-inheritance of the mitochondrial and W chromosomes immediately exposes incompatibilities between these maternal contributions of one species and the Z chromosome of another. We analyze mitochondrial and Z chromosome admixture in the long-tailed finch (Poephila acuticauda) of Australia, where hybridizing subspecies differ prominently in Z chromosome genotype and in bill color, yet the respective centers of geographic admixture for these two traits are offset by 350 km. We report two well-defined mitochondrial clades that diverged ∼0.5 million years ago. Mitochondrial contact is geographically co-located within a hybrid zone of Z chromosome admixture and is displaced from bill color admixture by nearly 400 km. Consistent with Haldane's rule expectations, hybrid zone females are significantly less likely than males to carry an admixed Z chromosome or have mismatched Z-mitochondrial genotypes. Furthermore, there are significantly fewer than expected mitonuclear mismatches in hybrid zone females and paternal backcross males. Results suggest a potential for mitonuclear/sex chromosome incompatibilities in the emergence of reproductive isolation in this system.

Hybrid sterility and evolution in Hawaiian Drosophila: differential gene and allele-specific expression analysis of backcross males.

The Hawaiian Drosophila are an iconic example of sequential colonization, adaptive radiation and speciation on islands. Genetic and phenotypic analysis of closely related species pairs that exhibit incomplete reproductive isolation can provide insights into the mechanisms of speciation. Drosophila silvestris from Hawai'i Island and Drosophila planitibia from Maui are two closely related allopatric Hawaiian picture-winged Drosophila that produce sterile F1 males but fertile F1 females, a pattern consistent with Haldane's rule. Backcrossing F1 hybrid females between these two species to parental species gives rise to recombinant males with three distinct sperm phenotypes despite a similar genomic background: motile sperm, no sperm (sterile), and immotile sperm. We found that these three reproductive morphologies of backcross hybrid males produce divergent gene expression profiles in testes, as measured with RNA sequencing. There were a total of 71 genes significantly differentially expressed between backcross males with no sperm compared with those backcross males with motile sperm and immotile sperm, but no significant differential gene expression between backcross males with motile sperm and backcross males with immotile sperm. All of these genes were underexpressed in males with no sperm, including a number of genes with previously known activities in adult testis. An allele-specific expression analysis showed overwhelmingly more cis-divergent than trans-divergent genes, with no significant difference in the ratio of cis- and trans-divergent genes among the sperm phenotypes. Overall, the results indicate that the regulation of gene expression involved in sperm production likely diverged relatively rapidly between these two closely related species.

A comparison of pollinator fig wasp development in figs of Ficus montana and its hybrids with Ficus asperifolia

AbstractFigs (Moraceae) and pollinator fig wasps (Hymenoptera: Agaonidae) have a highly specific mutualistic relationship but fig wasps occasionally enter atypical hosts, and this can lead to hybrid fig trees and the potential for gene flow between species. Many fig trees are dioecious, with fig wasp offspring developing in galled ovules inside figs on male trees, whereas seeds develop only in figs on female trees. We generated experimental hybrids between the Asian Ficus montana Blume and a closely related African species Ficus asperifolia Miquel. Male F1s were sterile if entered by Kradibia tentacularis (Grandi) (Agaonidae), the pollinator of F. montana, because its offspring always failed to develop, without ovule enlargement. As with the F1s, figs on most male backcross plants [F. montana × (F. montana × F. asperifolia)] also aborted shortly after pollinator entry, resulting in a higher turnover of figs than with F. montana, although the times taken for the figs to reach receptivity were similar. Pollinator larvae nonetheless consistently managed to develop inside the figs of one backcross plant and also occasionally in a few figs from another backcross individual. In these figs, galled ovules developed as normal, whereas in figs that aborted the galled ovules failed to enlarge. The sex ratio of K. tentacularis progeny in the backcross figs was female biased and did not differ from that in F. montana figs. Sycoscapter spec. (Hymenoptera: Pteromalidae), a parasitoid of K. tentacularis, was able to lay eggs and developed normally inside male backcross figs where its host was present.

Artificial interspecific backcrosses between the hybrid of female Crassostrea hongkongensis × male C. gigas and the two parental species

To explore the possibility of performance improvement in the hybrids of Crassostrea hongkongensis ♀×Crassostrea gigas ♂, backcross progeny and F2 hybrids were produced and evaluated. Three sets of replicates were successfully generated; each consisted of single crosses (HH, GG and HG), two female backcrosses between female hybrids and two male pure species (Hg/H and Hg/G), two male backcrosses between male hybrids and two female parents (H/Hg and G/Hg) and F2 hybrids (Hg/Hg). Fertilization, survival, growth and gonad development of these experimental groups were evaluated. All backcross groups exhibited high fertilization levels; however, the female backcrosses (Hg/H and Hg/G) and F2 hybrids exhibited a low hatching rate. Survival and growth superiority of the backcross progeny was observed from the larval stage to the adult stage, although outbreeding depression of the F2 hybrids occurred. Interestingly, all backcross progeny and F2 hybrids were fertile with functional gametes and normal sex ratios when fully matured. The genetic recombination of the backcross progeny and Mendel's genetic segregation of the F2 hybrids were detected with the nuclear gene marker in the backcross. Our results revealed that artificial interspecific backcrosses are successful, and the backcross progeny are viable, fertile, and fast-growing, and therefore hold promise for potential utilization in aquaculture as a new oyster stock resource. Statement of relevanceAll authors agree that the author list is correct in its content and order, and we have no conflict of interest to declare.

Haldane's rule is linked to extraordinary sex ratios and sperm length in stalk-eyed flies.

We use three allopatric populations of the stalk-eyed fly Teleopsis dalmanni from Southeast Asia to test two predictions made by the sex chromosome drive hypothesis for Haldane's rule. The first is that modifiers that suppress or enhance drive should evolve rapidly and independently in isolated populations. The second is that drive loci or modifiers should also cause sterility in hybrid males. We tested these predictions by assaying the fertility of 2066 males derived from backcross experiments involving two pairs of populations and found that the proportion of mated males that fail to produce any offspring ranged from 38 to 60% among crosses with some males producing strongly female-biased or male-biased sex ratios. After genotyping each male at 25-28 genetic markers we found quantitative trait loci (QTL) that jointly influence male sterility, sperm length, and biased progeny sex ratios in each pair of populations, but almost no shared QTL between population crosses. We also discovered that the extant X(SR) chromosome has no effect on sex ratio or sterility in these backcross males. Whether shared QTL are caused by linkage or pleiotropy requires additional study. Nevertheless, these results indicate the presence of a "cryptic" drive system that is currently masked by suppressing elements that are associated with sterility and sperm length within but not between populations and, therefore, must have evolved since the populations became isolated, i.e., in <100,000 years. We discuss how genes that influence sperm length may contribute to hybrid sterility.

X-y interactions underlie sperm head abnormality in hybrid male house mice.

The genetic basis of hybrid male sterility in house mice is complex, highly polygenic, and strongly X linked. Previous work suggested that there might be interactions between the Mus musculus musculus X and the M. m. domesticus Y with a large negative effect on sperm head morphology in hybrid males with an F1 autosomal background. To test this, we introgressed the M. m. domesticus Y onto a M. m. musculus background and measured the change in sperm morphology, testis weight, and sperm count across early backcross generations and in 11th generation backcross males in which the opportunity for X-autosome incompatibilities is effectively eliminated. We found that abnormality in sperm morphology persists in M. m. domesticus Y introgression males, and that this phenotype is rescued by M. m. domesticus introgressions on the X chromosome. In contrast, the severe reductions in testis weight and sperm count that characterize F1 males were eliminated after one generation of backcrossing. These results indicate that X-Y incompatibilities contribute specifically to sperm morphology. In contrast, X-autosome incompatibilities contribute to low testis weight, low sperm count, and sperm morphology. Restoration of normal testis weight and sperm count in first generation backcross males suggests that a small number of complex incompatibilities between loci on the M. m. musculus X and the M. m. domesticus autosomes underlie F1 male sterility. Together, these results provide insight into the genetic architecture of F1 male sterility and help to explain genome-wide patterns of introgression across the house mouse hybrid zone.

Sexual Selection, Epistasis and Species Boundaries in Sympatric Hawaiian Picture-winged Drosophila

The Hawaiian picture-winged flies in the genus Drosophila are a spectacular example of rapid evolutionary diversification in which sexual selection is considered an important mechanism for reproductive isolation and speciation. We investigated the behavioral reproductive isolation of two closely related and sympatric Hawaiian picture-winged Drosophila species, D. silvestris and D. heteroneura, which are known to hybridize in nature and produce viable and fertile hybrids. We compared the mating success of parental, F1 and backcross males in pairings with D. heteroneura females. The F1 males were produced by mating D. heteroneura males with D. silvestris females, and the backcross males were produced by mating F1 females with D. heteroneura males. The mating success of backcross males paired with D. heteroneura females were significantly reduced relative to that of parental and F1 males. This reduced mating success occurred primarily at a late stage of courtship where female choice of mate may be important. Two- and three-gene models demonstrate that epistasis involving a few genes could account for the observed variation in male mating success. These results are consistent with negative epistasis in the backcross generation and support the importance of sexual selection and negative epistasis in the evolution and maintenance of these species.

Ephemeral Association Between Gene CG5762 and Hybrid Male Sterility in Drosophila Sibling Species

Interspecies divergence in regulatory pathways may result in hybrid male sterility (HMS) when dominance and epistatic interactions between alleles that are functional within one genome are disrupted in hybrid genomes. The identification of genes contributing to HMS and other hybrid dysfunctions is essential for understanding the origin of new species (speciation). Previously, we identified a panel of male-specific loci misexpressed in sterile male hybrids of Drosophila simulans and D. mauritiana relative to parental species. In the current work, we attempt to dissect the genetic associations between HMS and one of the genes, CG5762, a Drosophila-unique locus characterized by rapid sequence divergence within the genus, presumably driven by positive natural selection. CG5762 is underexpressed in sterile backcross males compared with their fertile brothers. In CG5762 heterozygotes, the D. mauritiana allele is consistently overexpressed on both the D. simulans and D. mauritiana backcross genomic background, suggesting a cis-acting regulation factor. There is a significant association between heterozygosity and HMS in hybrid males from early but not later backcross generations. Microsatellite markers spanning CG5762 fail to associate with HMS. These observations lead to a conclusion that CG5762 is not a causative factor of HMS. Although genetic linkage between CG5762 and a neighboring causative introgression cannot be ruled out, it seems that the pattern is most consistent with CG5762 participating in epistatic interactions that are disrupted in flies with HMS.

Genetic analysis of maize cytoplasmic male sterile mutants obtained by space flight

Three maize male sterile mutants were obtained from the offsprings of two maize inbred lines 18-599 and 08-641, which were carried into space by the Shijian 8 Satellite. The stability of male sterile expression was observed in different locations, years, and seasons. In order to analyze the genetic characteristic of male sterility, testcross, backcross and reciprocal cross were made with these male sterile plants. The results showed that the male sterility character was stable in different locations, years, and seasons, and the sterility was inheritable. Because the maintainer lines and restorer lines for these sterile materials were found, and there was no male sterile plant separated among the reciprocal cross F2. Thus, we concluded that these mutants could be cytoplasmic male sterile. Combining the results of male fertility restoration test and PCR analysis, we could conclude that the three male sterile mutants were classified into the CMS-C type in maize. Owing to their difference in fertility restoration, these mutants may belong to different subgroups of CMS-C type. The discovery of the three male sterile mutants increased the genetic diversity of CMS-C type, improved the tolerance to Bipolaris maydis, and laid a foundation for extensive application of CMS-C in seeds production.

Inherited XX sex reversal originating from wild medaka populations

The teleost fish, medaka (Oryzias latipes), has an XX/XY sex-determining mechanism. A Y-linked DM domain gene, DMY, has been isolated by positional cloning as the sex-determining gene in this species. Previously, we conducted a field survey of genotypic sex and found that approximately 1% of wild medaka are sex-reversed (XX males and XY females). Here, we performed genetic analyses of nine spontaneous XX sex-reversed males to elucidate its genetic basis. In all cases, the F(1) progeny were all females, whereas XX males reappeared in the backcross (BC) progeny, suggesting that XX sex reversal is a recessive trait. Although the incidences of sex reversal in the BC progeny were mostly low, 40% were males derived from one XX male. We performed linkage analysis using 55 BC males and located a single major factor, sda-1 (sex-determining autosomal factor-1), controlling sex reversal in an autosomal linkage group. Thus, genes involved in the sex-determining pathway can be isolated from spontaneous mutants in wild populations.

Differences in DBA/1J and DBA/2J reveal lipid QTL genes

Recent advances in mouse genomics have revealed considerable variation in the form of single-nucleotide polymorphisms (SNPs) among common inbred strains. This has made it possible to characterize closely related strains and to identify genes that differ; such genes may be causal for quantitative phenotypes. The mouse strains DBA/1J and DBA/2J differ by just 5.6% at the SNP level. These strains exhibit differences in a number of metabolic and lipid phenotypes, such as plasma levels of triglycerides (TGs) and HDL. A cross between these strains revealed multiple quantitative trait loci (QTLs) in 294 progeny. We identified significant TG QTLs on chromosomes (Chrs) 1, 2, 3, 4, 8, 9, 10, 11, 12, 13, 14, 16, and 19, and significant HDL QTLs on Chrs 3, 9, and 16. Some QTLs mapped to chromosomes with limited variability between the two strains, thus facilitating the identification of candidate genes. We suggest that Tshr is the QTL gene for Chr 12 TG and HDL levels and that Ihh may account for the TG QTL on Chr 1. This cross highlights the advantage of crossing closely related strains for subsequent identification of QTL genes.

SSR based linkage and mapping analysis of C, a yellow cocoon gene in the silkworm, Bombyx mori

AbstractThe yellow color of the cocoon of the silkworm Bombyx mori is controlled by three genes, Y (Yellow haemolymph), I (Yellow inhibitor) and C (Outer‐layer yellow cocoon), which are located on linkage groups 2, 9 and 12, respectively. Taking advantage of a lack of crossing over in females, reciprocal backcrossed F1 (BC1) progeny were used for linkage analysis and mapping of the C gene using silkworm strains C108 and KY, which spin white and yellow cocoons, respectively. DNA was extracted from individual pupae and analyzed for simple sequence repeat (SSR) markers. The C gene was found to be linked to seven SSR markers. All the yellow cocoon individuals from a female heterozygous backcross (BC1 F) showed a heterozygous profile for SSR markers on linkage group 12, whereas individuals with light yellow cocoons showed the homozygous profile of the strain C108. Using a reciprocal heterozygous male backcross (BC1 M), we constructed a linkage map of 36.4 cM with the C gene located at the distal end, and the closest SSR marker at a distance of 13.9 cM.

Hybridization between wheat and jointed goatgrass (Aegilops cylindrica) under field conditions

Abstract Jointed goatgrass is an important weed of wheat in the United States and other parts of the world. Under field conditions, wheat and jointed goatgrass can hybridize and produce backcross derivatives, a situation that may allow gene flow between these two species. In order to gain a better understanding of the factors governing gene flow, a study to characterize patterns of mating between these two species was undertaken. Chloroplast and nuclear microsatellite markers were used to evaluate the parentage of 413 first-generation backcross (BC1) seeds obtained from 127 wheat–jointed goatgrass F1 hybrids, produced naturally under field conditions. Of the 127 hybrids evaluated, 109 (85.8%) had jointed goatgrass as the female parent, whereas the remaining 18 F1 plants (14.2%) had wheat as the female parent. Of the 413 BC1 plants analyzed, 358 (86.7%) had wheat and 24 (5.8%) had jointed goatgrass as the male backcross parent. The male parentage of 31 BC1 (7.5%) plants could not be determined. Under natur...

Influence of the hypogonadism (hgn) locus on female reproduction and ovarian development in an altered genetic background.

Background and Aims: The hypogonadic rat (hgn/hgn) shows male sterility controlled by a single recessive gene hgn. The hgn/hgn females detected by the presence of renal hypoplasia in the HGN inbred strain show a reduced fertility. Recently, the gene responsible for male hypogonadism was mapped on chromosome 10 by a linkage analysis using only male backcross progeny. Because backcross females could not be categorized as affected or normal because of variations in their renal sizes, we could not examine female fertility in the backcross progeny. In the present experiment, we examined whether the gene mapped on rat chromosome 10 has any influences on female reproduction and ovarian development. Methods: The assumptive hgn/hgn females were identified in the backcross progeny by microsatellite markers closely linked to the hgn locus. Postnatal body growth, the weights of reproductive organs, estrus cycle and ovarian histology were examined. In addition, backcross embryos were genotyped, and their body growth and ovarian development was examined. Results: The hgn/hgn females showed growth retardation, a short reproductive life and an abnormal ovarian histology as adults. Regarding embryonic development, hgn/hgn females showed body growth retardation and ovarian hypoplasia. Conclusion: The mutation of the hgn mapped on chromosome 10 causes not only male sterility but also female reduced fertility related to ovarian hypoplasia beyond the altered genetic background. (Reprod Med Biol 2006; 5: 227-234).

The genetic basis of postzygotic reproductive isolation between Drosophila santomea and D. yakuba due to hybrid male sterility.

A major unresolved challenge of evolutionary biology is to determine the nature of the allelic variants of "speciation genes": those alleles whose interaction produces inviable or infertile interspecific hybrids but does not reduce fitness in pure species. Here we map quantitative trait loci (QTL) affecting fertility of male hybrids between D. yakuba and its recently discovered sibling species, D. santomea. We mapped three to four X chromosome QTL and two autosomal QTL with large effects on the reduced fertility of D. yakuba and D. santomea backcross males. We observed epistasis between the X-linked QTL and also between the X and autosomal QTL. The X chromosome had a disproportionately large effect on hybrid sterility in both reciprocal backcross hybrids. However, the genetics of hybrid sterility differ between D. yakuba and D. santomea backcross males, both in terms of the magnitude of main effects and in the epistatic interactions. The QTL affecting hybrid fertility did not colocalize with QTL affecting sexual isolation in this species pair, but did colocalize with QTL affecting the marked difference in pigmentation between D. yakuba and D. santomea. These results provide the basis for future high-resolution mapping and ultimately, molecular cloning, of the interacting genes that contribute to hybrid sterility.

Timing of Development During Epiboly in Embryos of Second-Generation Crosses and Backcrosses between Odd- and Even-Broodyear Pink Salmon, Oncorhynchus gorbuscha

Odd- and even-year-spawning pink salmon (Oncorhynchus gorbuscha) are genetically isolated; their broodlines differ even in the same natal stream. Hybrids between broodlines exhibit outbreeding depression in survival. Variation in the time to completion of epiboly in embryos appears to be adaptive in both broodlines. We compared stage of development at a time near the completion of epiboly in families of second-generation offspring from crosses between odd- and even-year broodlines with development stages of within-broodyear controls and of backcrossed families. We observed embryos derived from matings of mature fish that were the results of fertilizations made 2 years earlier of eggs from females from the even brood year with semen from males from the even broodyear and with cryopreserved semen from males of the odd broodyear. The resulting fry had been released to the Pacific Ocean and recovered at maturity. Second generation embryos were produced by factorial matings of these mature fish involving (1) female and male controls, (2) female and male hybrids, and (3) both backcrosses. Analysis of variation of development time detected no effect of outbreeding, i.e., differences between controls and second generation hybrids (p > 0.05), but did detect variation between individual female parents (p < 0.03). Neither epistatic nor additive outbreeding depression could be detected in the rate of early embryonic development of pink salmon. However, effects on development rate attributable to female parents indicate that either a maternal effect or early additive genetic effects occurred before the expression of the paternal genome in embryos.

Reproductive isolation due to the genetic incompatibilities betweenThrichomys pachyurusand two subspecies ofThrichomys apereoides(Rodentia, Echimyidae)

We tested intrinsic reproductive isolation between 3 taxa of the South American caviomorph rodent Thrichomys (Rodentia, Echimyidae): T. pachyurus, T. apereoides subsp. apereoides and T. apereoides subsp. laurentius. They were mated in captivity and produced viable progeny. Some F1 hybrid females were fertile, whereas all F1 males were sterile. Histological examination revealed meiotic arrest at the primary spermatocyte stage. No sperm was detected in testes or epididymes. Electron microscopic analysis of surface spread synaptonemal complexes revealed a complete failure of chromosome pairing in F1 hybrids of T. pachyurus with T. apereoides subsp. laurentius and T. apereoides subsp. apereoides. In the male hybrids between T. apereoides subsp. apereoides and T. apereoides subsp. laurentius, meiosis did not proceed beyond diplotene, although all of the chromosomes, including heteromorphic ones, paired in an orderly fashion. Backcross males with homomorphic karyotypes showed segregation in meiosis progression. This indicates that male hybrid sterility is due to genetic, but not chromosomal, incompatibility of the parental taxa.

Hybrid origin of a swordtail species (Teleostei:Xiphophorus clemenciae) driven by sexual selection

The swordlike exaggerated caudal fin extensions of male swordtails are conspicuous traits that are selected for through female choice. Swords are one of only few examples where the hypothesis of a pre-existing bias is believed to apply for the evolution of a male trait. Previous laboratory experiments demonstrated that females prefer males with longer swords and even females from some swordless species show an affiliation for males of sworded species. Earlier phylogenetic studies based on maternally inherited mitochondrial DNA placed the sworded southern swordtail Xiphophorus clemenciae with swordless platies, contradicting its morphology-based evolutionary affinities. The analyses of new nuclear DNA markers now recover its traditional phylogenetic placement with other southern swordtails, suggesting that this species was formed by an ancient hybridization event. We propose that sexual selection through female choice was the likely process of hybrid speciation, by mating of platy females with males of an ancestral swordtail lineage. In artificial crosses of descendent species from the two potential ancestral lineages of X. clemenciae the hybrid and backcross males have swords of intermediate lengths. Additionally, mate choice experiments demonstrate that hybrid females prefer sworded males. These experimental lines of evidence make hybridization through xeno-specific sexual selection by female choice the likely mechanism of speciation.

Genetic interactions underlying hybrid male sterility in the Drosophila bipectinata species complex

Understanding genetic mechanisms underlying hybrid male sterility is one of the most challenging problems in evolutionary biology especially speciation. By using the interspecific hybridization method roles of Y chromosome, Major Hybrid Sterility (MHS) genes and cytoplasm in sterility of hybrid males have been investigated in a promising group, the Drosophila bipectinata species complex that consists of four closely related species: D. pseudoananassae, D. bipectinata, D. parabipectinata and D. malerkotliana. The interspecific introgression analyses show that neither cytoplasm nor MHS genes are involved but X-Y interactions may be playing major role in hybrid male sterility between D. pseudoananassae and the other three species. The results of interspecific introgression analyses also show considerable decrease in the number of males in the backcross offspring and all males have atrophied testes. There is a significant positive correlation between sex - ratio distortion and severity of sterility in backcross males. These findings provide evidence that D. pseudoananassae is remotely related with other three species of the D. bipectinata species complex.