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Geographic patterns of koala retrovirus genetic diversity, endogenization and subtype distributions

Koala retrovirus subtype A is the youngest endogenized retrovirus, providing a unique system to elucidate retroviral-host co-evolution. We characterised KoRV geography using faecal DNA from 192 samples across 20 populations throughout the koala’s range. We reveal an abrupt change in KoRV genetics and incidence at the Victoria/NSW state border. In northern koalas, pol gene copies were ubiquitously present at greater than 5 per-cell, consistent with endogenous KoRV. In southern koalas, pol copies were detected in only 25.8% of koalas and always at copy numbers less than one, while the env gene was detected in all animals and in a majority at copy numbers of greater than one per-cell. These results suggest that southern koalas carry partial endogenous KoRV-like sequences. Deep sequencing of the env hypervariable region revealed three putatively endogenous KoRV-A sequences in northern koalas and a single, distinct sequence present in all southern koalas. Among northern populations, env sequence diversity decreased with distance from the equator, suggesting infectious KoRV-A invaded the koala genome in northern Australia and then spread south. The previously described exogenous KoRV subtypes (B-K), two novel subtypes (L and M), and intermediate or hybrid subtypes were detected in all northern koala populations but strikingly absent from all southern animals tested. Apart from KoRV-D, these exogenous subtypes were generally locally prevalent but geographically restricted, producing KoRV genetic differentiation among northern populations. This suggests that sporadic evolution and local transmission of the exogenous subtypes has occurred within northern Australia, but this has not extended into animals within southern Australia. Author Summary Retrovirus infection is generally synonymous with disease; however, retroviruses can also become endogenous (incorporated into the germline) and thereby directly contribute to the genetic makeup of a species. This has occurred in all vertebrates, yet little is known about the endogenization process. As the youngest virus known to be endogenized, koala retrovirus (KoRV) offers a unique opportunity to study these early stages of co-evolution. This study reveals a comprehensive picture of KoRV biogeography that informs our understanding of how host population history, host suppression and transmission dynamics can influence retroviral evolution. KoRV is also associated with chlamydiosis and neoplasia in the vulnerable koala. Our improved understanding of how KoRV variants are distributed should guide conservation management to help limit disease.

146 cell as occurs when endogenous sequences are present. This strongly suggests that intact KoRV is 147 not endogenized in those populations. However, it should be noted that these copy numbers are based 148 on an estimate of 14 copies of β-actin in the koala diploid genome. If the actual number of β-actin 149 copies was in fact 16 then the KoRV pol copy number in one of the Ulupna koalas would exceed one.
150 Therefore, the presence of intact endogenous KoRV at low incidence should not be discounted for 151 the Ulupna population.

152
In stark contrast to the findings for KoRV pol, the incidence of KoRV-A env among the 97 153 southern koalas was 100% as determined by qPCR. Of these koalas 35 produced melt curve peaks at 154 multiple temperatures and 11 did not have a peak at the expected temperature for the original KoRV-A 155 env fragment. This potentially indicates that these samples contained multiple sequence variants, 156 preventing accurate copy number estimation. Of the remaining 62 samples, 46 (74.2 %) had greater 157 than 1 env copy per cell (mean = 11.2, range = 1.1 -56.3, excluding an outlier from Bessiebelle that 158 had 209 copies per cell) ( Fig 1D). This suggests that at least a fragment of the KoRV env gene is 159 endogenous in the majority of southern koalas.  (Table 2). Among these sequences, 384 were detected in northern koalas and 116 were detected in 228 southern koalas. Similarly, among the 313 intact sequences there were far more identified in northern 229 koalas (279 sequence clusters) than among southern koalas (41 sequence clusters) with only a very 230 small number (7 sequence clusters) shared between the north and the south ( Table 2). This was not 231 an effect of differing sample sizes between regions as shown by rarefaction (Fig. 4D).

232
At the population level, there was considerably less sequence cluster sharing between northern 233 and southern populations than among populations within those regions (average number of shared 234 sequence clusters between two populations: north vs south = 1.57, north vs north = 6.78, south vs 235 south = 7.68; Fig 4A). However, within the north or south there was no evidence that populations that 236 were geographically closer together shared more sequence clusters (Mantel test with significance 237 determined by permutation, north: p = 0.18; south: p = 0.13).

238
The number of intact sequences found within populations for the same number of sampled 239 koalas was generally similar in the north and south (Fig. 4G). This suggests that the high total number 240 of unique sequences in the north is due to differences between populations in the sequences found, 241 rather than higher within population richness (Table 2). An exception to this general finding is the 242 Monaro population (NSW), where a higher number of sequence clusters were identified with each 243 additional koala sampled than for the other populations ( Fig 4G).

244
The patterns of intact KoRV-A sequence sharing among populations reflected those seen for 245 all sequences combined, with less sharing between the north and the south than within those regions 246 (Table 2, Fig. 4B). However, in contrast to the patterns observed for all intact sequences combined, 247 a similar number of unique KoRV-A sequence clusters were identified among southern (n =38) and 248 northern (n = 40) populations, despite fewer koalas being sampled in the south (Fig. 4E). This 249 indicates that each southern koala carries more unique KoRV-A sequences than do northern koalas 250 and this is reflected in a higher number of unique sequences being identified per southern population, 251 after accounting for differences in sample sizes (Fig. 4H). Again, the Monaro population was found 252 to be an exception and had similar KoRV-A richness to the southern populations ( Fig 4H). 258 env genetic diversity throughout the koala range 259 In order to assess genetic diversity in the env sequence without the added complexity of subtype 260 presence, the pairwise number of nucleotide differences between sequences was calculated, excluding 261 the hypervariable region (that is likely to be derived through recombination and thereby does not 262 adhere to stepwise diversification). In general, the mean number of nucleotide differences (MND) 263 among all pairs of intact and non-functional sequence clusters detected within a population was 264 significantly higher for the northern populations than for the southern populations, although not by a 265 large margin (mean ± SD: north = 17.35 ± 3.03, south = 13.13 ± 2.83; p =0.007; Fig 5A).

266
There was no distinct geographic pattern in sequence diversity for southern populations (  Table 3). There was no differentiation between southern 289 populations (variance attributed to differences between southern populations = 0 %; p = 0.755), while 290 there was significant differentiation accounting for 8% of the total genetic variance across northern 291 populations (p =0.001; Table 3). How different the northern populations were from one another varied 292 between pairs of northern populations (Table S1), however, populations that were geographically 293 more distant were not more genetically different (Mantel test between geographic distance and PhiPT: 294 p = 0.208; Fig S5). Instead, the diversity of KoRV sequences within each northern population was 295 distinct (Table S1), with the Monaro considerably more distinct than the other populations (average 296 pairwise PhiPT: Monaro = 0.14; other populations = 0.03 to 0.08).

297
When considering the different types of sequence clusters separately, KoRV-A differentiation 298 was non-significant among northern and southern populations, while there was a low but significant 299 level of differentiation among regions (Table 3). There was also no differentiation among southern 300 populations for the non-functional sequence clusters, while there was low but significant 301 differentiation among northern populations (Table 3). By contrast, there was considerable 302 differentiation in KoRV-B to M subtype sequence clusters between northern populations (despite the 303 exclusion of the hypervariable region from the analysis; variance attributed = 19%; p < 0.001; Table   304 3). Thus, the total env genetic differentiation observed between northern populations can primarily 305 be attributed to KoRV-B to M.

The effect of population incidence and within koala abundance on genetic differentiation
307 When the incidence of (number of koalas carrying) the sequence clusters within each population was 308 included in the AMOVA analysis (instead of only their presence), overall differentiation among 309 northern populations increased slightly from 8% to 10%, while very low but significant differentiation 310 was found among southern populations (Table 3). Further, when the sequences were also weighted 311 by their average within koala abundance in the AMOVA, differentiation among northern populations 312 increased markedly to 26%, while there remained no differentiation among southern populations 313 (Table 3). This marked increase in differentiation among northern populations reflects the trend for 314 the more abundant sequence clusters within koalas to be found in fewer populations (regression z 315 value = -1.753, p = 0.080; Fig S6b).

316
Among sequence clusters detected in northern koalas, those that had a high incidence within 317 a population were also found at higher abundance within koalas from that population (regression z 318 value = 3.277, p = 0.001 Fig S6a). Therefore, between-koala differences in KoRV env sequence 319 composition that accounted for 7% of genetic variation based on incidence (p= 0.001) became non-320 significant when within koala abundance was taken into account (genetic variance = 1%, p =0.554;