The Spirostreptida order of millipedes is exceptionally abundant and widely distributed across Africa, encompassing approximately 71 recorded genera. Extensive research on spirostreptid millipedes has revealed significant genetic divergence among these organisms, with an average pairwise distance of 10.7% observed (Hamer 1999, Mwabvu et al. 2015). Among the numerous millipede genera in Africa, Bicoxidens stands out as one of the most extensively studied. This genus is exclusively endemic to the southern regions of the continent and thrives in diverse habitats such as savanna woodlands, forests, and riverine vegetation (Mwabvu et al.2009, Mwabvu et al., 2010, Mwabvu et al. 2015). Bicoxidens predominantly occur in Zimbabwe's eastern, southern, and central areas, as documented by Mwabvu et al. (2009). Nine distinct species have been identified within this genus, with Bicoxidens flavicollis exhibiting noteworthy phenotypic variation across different populations (Tinago et al. 2017).
In a comprehensive study conducted by Tinago et al. et al.(2017), mitochondrial DNA sequences were employed to investigate the genetic diversity of Bicoxidens populations in Zimbabwe. The findings unveiled the existence of several distinct mitochondrial lineages within the genus, indicating potential geographic isolation and/or ecological adaptation as contributing factors to their divergence. These multiple lineages strongly suggest the presence of cryptic species concealed within the genus Bicoxidens. The research results highlighted the presence of numerous divergent lineages within the Bicoxidens genus. Remarkably, some lineages were found to be more closely related to each other than to other lineages within the same genus. Such a pattern indicates prolonged geographic isolation among the various populations of Bicoxidens, suggesting that these lineages have been evolving independently for an extended period (Tinago et al 2017).
A molecular phylogenetic analysis of the millipede family Spirostreptidae in Africa revealed a complex pattern of diversification and radiation. Several genera within the Spirostreptida have undergone thorough revision, shedding light on their taxonomy and characteristics. Among these revised genera are Archispirostreptus, Doratogonus, Cacuminostreptus, Spirostreptus, Plagiotaphrus, and Bicoxidens, as documented by (Hamer 2000, Mwabvuet al. 2009, Mwabvu 2010, Mwabvu et al. 2015). The millipede fauna of North Africa has been relatively well-studied, with the Julida order being the richest and most diverse group (Akkar et al 2009). This order encompasses 58 species distributed among 12 genera and 3 families, and is predominantly found in Algeria, Egypt, Morocco, Tunisia, and Libya (Akkaret al. 2009). In contrast, the millipede fauna of Sub-Saharan Africa is less well-studied, with a considerable gap in our knowledge. However, the region hosts a remarkably rich diplopod fauna, boasting approximately 552 species belonging to 71 genera. Among these, the Spirostreptida order is the most recognizable, conspicuous, and frequently encountered group of diplopods in urban areas of Southern Africa (Hamer 1999).
Mitochondrial DNA (mtDNA) is a frequently utilized tool in genetic research due to its haploid nature, which facilitates its amplification across diverse taxa without the need for cloning procedures (Hurst and Jiggins 2005). mtDNA is also valuable for researchers because its structure and sequence can provide insights into evolution, gene flow, phylogenetics, and molecular evolution (Mandal et al. 2014). Despite constituting only a small fraction of an organism's entire genome, mtDNA remains the most commonly employed marker for studying molecular genetic diversity (Galtier et al 2009).
Ribosomal RNA (rRNA) genes have gained significant importance in phylogenetic studies because they have a high gene copy number per cell, which facilitates efficient gene amplification and sequencing (Galtier et al 2009). These genes contain conserved regions and variable regions, including expansion segments, which provide valuable information on evolutionary rates of base substitutions within the rRNA gene (Gillespieet al. 2006). Specifically, the 16S rRNA gene, a component of the ribosomal subunit, plays a crucial role in phylogenetic analysis. Its conserved secondary structure, in conjunction with associated proteins, forms the large mitochondrial RNA sub-unit (Schubart, et al. 2000).
The 16S rRNA region is a valuable tool for studying interspecific differentiation because it exhibits relatively low evolutionary rates, meaning that it changes slowly over time (Calo–Mata et al. 2009). This makes it ideal for comparing the genetic sequences of different species, even if they are closely related. The presence of conserved and variable regions within the same gene also makes 16S rRNA a favored marker for investigating separation events and conducting phylogenetic reconstructions (Schubart et al. 2000). In many phylogenetic studies involving diverse species, the genes 12S and 16S rRNA are commonly employed in conjunction with each other, as this provides a more complete picture of the evolutionary relationships between the species (Kuznetsova et al, 2002). The highly accelerated evolutionary rate of mitochondrial DNA (mtDNA) allows for the observation of substantial variation between closely related species (Yang and Bielawski 2000).
Microsatellites are widely dispersed throughout the genome and consist of short tandem repeat sequences, such as di-, tri-, or tetranucleotide repeats, with variable lengths ranging from one to five base pairs (Abdul Muneer 2014).This unique feature makes microsatellites a popular choice for molecular studies and the assessment of genetic population structures (Richard and Thorpe 2001, Abdul Muneer 2014). While most population genetic studies rely on data obtained from mitochondrial and nuclear markers, the information derived from evaluating microsatellites is crucial (Abdul Muneer 2014). Microsatellite sequences can be classified into minisatellites, and microsatellites based on their size. Microsatellites are particularly useful in identifying closely related populations due to their high variability levels, ability to isolate numerous loci, and fast processing speed (Abdul Muneer 2014).
Microsatellites are abundant in eukaryotic genomes, and their alleles are inherited according to Mendelian genetics. Each microsatellite locus is characterized by a known DNA sequence, consisting of both unique and repetitive DNA segments (Richard and Thorpe 2001, Abdul Muneer 2014). In a study by (Wojcieszek and Simmons 2009), 25 novel microsatellite markers were isolated from the millipede species Antichiropus variables to investigate patterns of paternity. These markers represented the first microsatellite loci identified in a millipede species. Among the 25 loci, eleven were found to be polymorphic, and eight loci successfully amplified in other species of Antichiropus (Wojcieszek. and Simmons 2009). Similarly, Hasegawa et al. (2011) examined thirteen newly isolated polymorphic microsatellite loci in Brachycybe nodulosa. Out of these thirteen loci, only two showed lower-than-expected heterozygosity. These new loci hold potential for conservation efforts not only for Brachycybe nodulosa but also for other species within the same taxonomic group (Hasegawa et al. 2011). A study by Marek et al. (2012) examined the genetic diversity of millipedes in the family Polydesmida in the United States. The study used a combination of molecular markers and morphological traits to analyze the genetic relationships among different species and populations and found high levels of genetic diversity within and among populations. The researchers suggested that the complex geological history and biogeographical patterns of the United States may have influenced the evolution and diversification of millipedes in this region.
A study conducted by Marek et al. (2012) that was used DNA sequencing to identify several distinct genetic lineages within the species (Pachybolus ligulatus) and suggested that this diversity may be linked to differences in environmental conditions such as temperature and rainfall. They also found evidence of limited gene flow between populations, which could have implications for the species' ability to adapt to changing environmental conditions. A study by Vladimír et al. ( 2013) used genotyping-by-sequencing to investigate the genetic diversity and population structure of the millipede species Narceus americanus in the southeastern United States. The researchers found evidence of high genetic diversity within populations, with limited gene flow between populations. Overall, these studies suggest that millipedes exhibit high levels of genetic diversity, both within and among populations. The specific patterns of genetic diversity may be influenced by a variety of factors, including historical climate change, landscape evolution, and biogeographical patterns.