Psammotermes allocerus, Silvestri, 1908, Silvestri, 1908

Gunter, Felicitas, Oldeland, Jens, Picker, Mike D., Henschel, Joh R. & Jürgens, Norbert, 2023, Cryptic subterranean diversity: regional phylogeography of the sand termite Psammotermes allocerus Silvestri, 1908 in the wider Namib region, Organisms Diversity & Evolution 23 (1), pp. 139-150 : 141-142

Results

Phylogeny analyses

The mitochondrial sequences were visually examined according to the presence of NUMTs %nuclear mitochondrial sequences). Characteristics such as stop codons, frameshift mutations and insertions-deletions were not detected. Finally, the COI marker %362 bp) contained 137 constant, 70 uninformative, and 155 parsimony-informative positions. For the second marker, COII %448 bp), 259 positions were constant, 89 were uninformative, and 124 were parsimony-informative. The concatenated alignment %810 bp) resulted in 507 constant positions. A low number of 92 positions were uninformative, and 211 were parsimony-informative. All mitochondrial COI and COII sequences were concatenated in one alignment, and the resulting Bayesian Inference %BI) analysis shows a strongly supported differentiation of the 65 P. allocerus samples from the outgroup %1 posterior probabilities %pp)). We identified seven genetic groups which may represent putative species clusters according to the high posterior probability values of the nodes %pp> 0.95), higher values of p-distances between these clusters as within the clusters as well haplotype analyses according to Roy et al. %2006) and Hausberger et al. %2011). The seven strongly supported clades %0.99 pp, Fig. 1 ), roughly fit a differentiation from south to north within the study area % Fig. 2 ).

The most basal clade within Psammotermes %dark blue, Fig. 1 ) was the ‘Succulent Karoo’ clade, as all seven samples are located within the Succulent Karoo biome in the north-western part of the winter rainfall region of South Africa % Fig. 2 ). It included the collection of the topotype near Lüderitz %LUE, Namibia). The next clearly distinguishable genetic group was the ‘ Southern Namib’ clade, with the majority of the samples located at the eastern margin of Southern Namib % Fig. 2 ). Three localities are very well supported %> 0.99 pp), and only two are sisters to one other %RO and DV). The next higher positioned clade was split into two larger subclades at an equal level of differentiation, one occurring in south-eastern Namibia, adjacent to South Africa. The other clade includes samples from northern Namibia and Angola. The well-supported subclade % Fig. 1 ) includes collections from south-eastern Namibia and the eastern Richtersveld % South Africa). Phylogenetically it is subdivided into three genetic groups: ‘ East Gariep’ , ‘ Southwestern Kalahari’ and ‘ Nama’ . The ‘ East Gariep’ group is represented by 13 sites, of which six are located in the northeastern Richtersveld and the adjacent larger Orange River valley in Namibia % Fig. 2 ). The weakly supported %0.52 pp) combined groups ‘ Nama’ and ‘ Southwestern Kalahari’ are sisters to the ‘ East Gariep’ group. Both individual groups % Southwestern Kalahari and Nama ) are very well supported %1 pp). The ‘ Southwestern Kalahari’ clade comprises eight collection sites situated in dunes and sand valleys east of the Karas mountains. The ‘ Nama’ group %yellow) includes six study sites in the southern Namaland of the Namibian Karas Region. The second larger subclade is well supported %0.92 pp) and included the groups ‘ Western Kalahari Basin’ and ‘ Northern Namib’ , both very well supported %1 pp). The ‘ Western Kalahari Basin’ comprises 11 collection sites ordered in a polytomy. This group includes sites that are located from south-eastern Namibia %SK3, SA) to the Waterberg %RI and RV) and the north-easternmost site Katima Mulilo %KM). The ‘ Northern Namib’ group encompassed all remaining study sites, located from the Rössing mountain %ROE) to the Iona National Park in Angola %IO).

Overall, the mean p -distances are higher between the seven groups than within each group % Tables 1 and 2 ). The mean p -distance between the groups ranges from 0.0508 %‘Nama’ vs. ‘East Gariep’) to 0.1007 %‘Kalahari Basin’ vs. ‘Succulent Karoo’). The values of the mean p -distance are lower and ranged from 0.0100 %‘Succulent Karoo’) to 0.0363 %‘Northern Namib’).

Haplotype analyses

The median-joining network based on the concatenated alignment comprised 31 observed haplotypes, including 18 unique haplotypes %which includes a single sample) and 12 calculated hypothetical haplotypes % Fig. 3 ). Eight hypothetical haplotypes are situated in the centre of the median-joining network. The observed haplotypes of the seven genetic groups are linked over this centre. The highest value of unique haplotypes was calculated for the ‘Southern Namib’ group with six haplotypes. In contrast, the ‘Succulent Karoo’ comprised two haplotypes calculated for six sequences. The two groups, ‘Western Kalahari Basin’ and ‘Succulent Karoo’, are connected to the centre with a high number of mutation steps %8 and 10, Fig. 3 ). The number of haplotypes differed slightly between the median-joining network analysis % Fig. 3 ) and the calculated number of haplotypes gained by DnaSP v6 % Table 3 ). The nucleotide diversity %π) ranged from 0.00416 %‘Western Kalahari Basin’) to 0.01597 %‘Northern Namib’, Table 3 ). The lowest haplotype diversity was calculated for the ‘Succulent Karoo’ with 0.78571 and the highest for the ‘Southern Namib’ with 1.0000 % Table 3 ). The number of segregating sites %S) was highest within the ‘Southern Namib’ group with 22 S and lowest within the ‘Succulent Karoo’ and ‘Western Kalahari Basin’ with each six S % Table 3 ). The genetic groups undergo a different selection pressure. A positive Tajima’s D value was calculated for the groups: ‘Northern Namib’, ‘Western Kalahari Basin’ and ‘Southwestern Kalahari’ and a negative Tajima’s D value was estimated for the remaining groups %‘Nama’, ‘Southern Namib’, ‘East Gariep’, ‘Succulent Karoo’, Table 3 ).

Two additional features distinguish the ‘Succulent Karoo’ group from the other groups. The tapetum of the tunnels within the nest system of P. allocerus colonies in the ‘Succulent Karoo’ is whitish. In all other groups, it is blackish % Fig. 4 ). Only in the ‘Succulent Karoo’ nests was it possible to locate the royal chambers with paired kings and queens at a depth of 15 to 30 cm below the soil surface % Fig. 4 ). In all other groups, one of the authors %NJ) never locate royal chambers or kings or queens in the first 100 cm below the soil surface, despite some 100 nest excavations.

Figures

Fig. 1 Phylogeny of 65 P. allocerus collections inferred by the Bayesian analysis of COI and COII markers. Support values are given in posterior probability. Clades are coloured according to the genetic group. Dark blue: Succulent Karoo; Light Blue: Southern Namib; Dark green: East Gariep; Light Green: Southwestern Kalahari; Yellow: Nama; Ochre: Western Kalahari Basin; Red: Northern Namib. Abbreviations of study sites are shown in Fig. 2

Fig.2 Distribution map of all Psammotermes allocerus collections and their assignment to the genetic groups found in the phylogenetic analyses %colours). Circles: Collections used for the phylogeny. Black lines: Country borders. Northern Namib: %AH) Aba Huab rivier, %GV) Giribesvlakte, %HD) Hartmann Dunes, %HO) Hoada, %IO) Iona, %MF) Marienfluss, %PR) Purros, %ROE) Rössing mountain, %SO) Sorris Sorris, %TM) Tomakas; Western Kalahari Basin: %AHV) Alt-Hartebeestvlei, %AM) Alex Muranda, %GU) Gariganus, %KM) Katima Mulilo, %RI) Rimini, %RS) Rundu South, %RV) Ravenna, %SA) Samehaling, %SK) Swartkop, %WF2) Warmfontein; Nama: %BY) Barby, %FRC) Fish river Canyon, %GO) Goageb, %SH) Seeheim, %WV) Witvley; Southwestern

Fig. 3 Median-joining network of the combined COI and COII P. allocerus sequences. Coloured circles represent the observed haplotypes, and the size is proportional to the num- ber of collections. Black circles represent missing haplotypes. Marks show the number of mutation steps. Haplotype num- bers are gained from DnaSP v6. Dotted lines and colours mark haplotypes according to the genetic group of the phylogeny

Fig.4 Differences in the tape- tum colour of three P. allocerus colonies and the royal pair. A Whitish tapetum of a nest from the Springklipplain from the ‘Succulent Karoo’ group %South Africa, 26 September 2016). B First image of the king and queen of P. allocerus from Yellow Dune %‘Succulent Karoo’, South Africa, 08 March 2015). C Blackish tapetum of a nest from Dieprivier %‘Southern Namib’, Namibia, 05 April 2017). D Blackish tapetum and chambers filled with foraged grass from Iona %‘Northern Namib’, Angola, 26 September 2016). Images taken by Norbert Jürgens, Felicitas Gunter