Cryptic New Guinean mammals

Good morning, thank you for the opportunity to present here my project.

I come from the Czech Republic which is a small country in the middle of Central Europe; and more specifically, I'm based at the University of Southern Bohemia in České Budějovice. I'm employed at the Biology Centre of the Czech Academy of Sciences in the same town. Why have I decided to study the elevational distribution in non-tropical non-volant mammals? Well, first, why non volant or non-flying mammals, it's because their dispersal abilities are very limited because of the combination of two factors.

First, they’re flightless, so they have difficulties in going across large water bodies, steep mountains, valleys. And second, they are endothermic; they are warm bodied, so they require a constant intake of food. And the combination of these two factors makes them actually very poor migrators. Next, the tropical rainforests are well known to be the most diverse tropical terrestrial ecosystem. And also in combination with high topographical complexity, which means very steep and high mountains, they present very fertile ground for biodiversity, for speciation in vertebrates. And all of these things are interconnected together with the other topic that I'm really focused on. And that's the elevational gradient which is one of the basic pillars of ecology itself because it's important for many reasons. For example, it enables us to study the community ecology, the interactions and requirements of the different species and communities and populations that differ a lot in their requirements, but they're found within a very small area because the different altitudes present different temperatures, different microhabitats, and correspondingly different sources which is very interesting.

This topic, the elevational distribution in tropical flightless mammals, was, as I will show you in a moment, studied across many countries and tropical continents. However, the results still differ, we still don't actually know what's precisely going on. So it is very important, from my point of view, and very interesting. And that's the reason why I've decided to go into this. Here you can see that basically all continents, or should I say, zoogeographical realms, were studied, except for the Oceanian region, or, more specifically, the Australo-Oceanian because the only study that was conducted in Australian Tropics on non-volant mammals was on Mount Lewis which is around 1000 meters tall which, compared to the other continents, is really not comparable. So it's necessary to really focus on Oceania because, for example, the island of New Guinea has the Central Highlands, that are covered with really high mountains. In modern history, there was one study that really focused on the elevation gradient of comparable size and sampling, and it was the study in the 1980s conducted in West Sepik which is now Sandaun province in the west of Eastern Papua New Guinea. So I decided to go into this more thoroughly and deeply.

For my study site, I've chosen the highest mountain of New Guinea, I mean Papua New Guinea and actually the whole Oceania, which is Mount Wilhelm. Conveniently, there is established the altitudinal gradient by Professor Vojtech Novotny that Kris already mentioned, who for more than 20 years now, is a leader of New Guinea Binatang Research Centre based near Madang. They have already studied many taxa along this gradient, such as plants, insects, and different vertebrates, but non-volant mammals are still missing. For obvious reasons, they are nocturnal, they don't vocalise, so they are very difficult to be actually seen, or tracked. Regarding the elevations, they extend from the lowland at 200 metres up to 3700 metres above sea level by 500 elevational metres. And additionally, I've added one more locality at just the sea site close to the Madang.

So, in 2019, I spent six months sampling the elevational gradient. You can see that the localities at each elevation look very different, the environment at each altitude is very different. And you may imagine that spending quite a long time in such an adventurous country as Papua isalso brings some adventurous experience. Some of them might be considered dangerous, but I made through and I've enjoyed myself a lot. I focused mostly on trapping, I was using snap traps, live traps, both boxes and cages and also pitfall trapping which, if I'm correct, was the first time using this methodology in Papua New Guinea. And it was very successful. I was setting the traps in lines trying to cover as many different habitats at each locality as possible, such as primary forest, secondary forest, gardens, huts, grassland, water currents, also ecotones, and partly canopy as well. And then, I've tried also different techniques to record as many species as possible present at each locality, such as collecting skins and skulls which are trophies, using some camera traps, spotlighting, and mostly cooperating with local people because they know their animals the most and the best. I was doing plenty of interviews, but most of all, I was joining the hunters on their hunts, going and trying to go to the jungle at night as many times as possible, and it was great.

So, after six months, the longest elevational gradient in tropics was done. I finally finalised the expedition, the study, but we decided that this is not enough and we want to continue. So the next year, a Papuan colleague of mine, Daniel Okena, who is actually now starting his PhD in Czech Republic, repeated my protocol in the Finisterre Range. Five localities are already sampled with three more going to be sampled within the next two months now. Why did we choose this Finisterre Range that's located on the Huon peninsula in the east of New Guinea? Well, from the zoogeographical point of view, it's a very fascinating place because it lies just on the border where two tectonic plates are subducting one under the other. The Australian one in the south is touching the Pacific plate in the north. And both of the mountain ranges are located at different plates. As you can see in the picture in the left, they are of similar height reaching up to 4000 metres, and at the very closest distance, they are only 9 kilometres apart, so they're very close to each other. However, in between there is a huge lowland valley filled with two great rivers, and also very distinctive lowland vegetation just on the place where the two plates touch, and also, they are of a very different age, because the Central Highlands, where the Mount Wilhelm is located, is about 30 million years old, because it's of the old Australian Craton origin. While on the other hand, the Finisterre Range is only 5 million years old. So you can imagine how steep it is compared to the to the Mount Wilhelm. And since the last rodents arrived to New Guinea around 7 million years ago, it's very close to the age of origin of the of the Finisterre Range. And I'm very curious of comparing these similar mountain ranges located very close to each other, but having a totally different origin and age.

In the table here, you can see some results of previous similar studies in other tropical countries, Philippines, the one from 1980s that I've mentioned at the very beginning, and combined results of our expeditions in 2019 and the five localities from 2020. In the trapnight column, you can basically see the sampling effort, which is a combination of number of traps and number of trapping days. And then the following numbers are stating number of individuals that were trapped, and number of species recorded. Based on this, I could do the preliminary, well, the first ecological analysis, which already by itself show very interesting results. I’ve noticed that all of the previous expeditions, or all of the previous studies, were based only on in-field morphological determination of species. So I got a severe suspicion because, as you know, most of the, well, a lot of rodents look the same. They are small and dully coloured, so it's difficult to tell apart the species, and I suspect that there is a huge, there might be a huge crypticity among the species, meaning that what we think is one single species, may be actually more of them.

So that's the reason why I've decided to go into the molecular barcoding which means determining the species by the means of genetics. Altogether from the two years of expeditions, we have about 1000 samples because I decided to sample every single individual that was collected alongside the two gradients. That's a lot. So I started cooperation with my colleague from Southern France, Dr. Pierre- Henri Fabre, from the Institute of Evolutionary sciences in Montpellier. They have great equipment in their molecular labs. We decided to start with three genes, both mitochondrial and nuclear. To be able to compare the species, we needed to create our own library first. And for this, we needed to design our own primers and protocols. We succeeded in this, so now we have a library and primers, which are specific for each genus of the New Guinean rodents and marsupial's that I brought. Next, I could continue with the PCR. So I've conducted around 2000 PCR reactions to amplify the focal genes, then we could read them. And after this was done, I counted the species delimitation. In mammals, it's usually said that the species diverge or differ by 1%. If they differ by 1% of their genetic information, they are considered to be distinctive species. To be more conservative, and to avoid over splitting of some species, I've decided to go for 5%. And with all of this, I was able to construct my first phylogenetic trees of life.

And these showed us some first results. So first of all, yes, it was confirmed that there is a rather large crypticity among the non-volant New Guinean mammals. So what we thought is just one species is actually many of them, which actually shows us that the biodiversity of non-volant mammals in New Guinea is probably much higher than we thought it is. And then, every individual was genetically determined along the gradients which is, a far as I know, the first time that this was done for tropical non-volant mammals. Well, I said, the species are largely cryptic, but in fact, when we focused on the groups divided by the genetic results, we are noticing that they actually have some morphological features, some morphological characteristics, according to which they can be divided. So they are not actually really cryptic, unrecognisable, but more likely, the species diversity was overlooked because of these kind of characteristics. And that's what brought me here to the Australian Museum. I'm lucky to cooperate with Professor Kris Helgen, and to gain access to the wonderful collections in this museum, which were largely made also by Tim Flannery. So here, I'm documenting and measuring both the external and bone characteristics.

After this will be done – as Kris already mentioned, we will move to the other museums. We are already cooperating with the relevant European museums, and after I finish here, we will continue to the National Museum of Papua New Guinea in Port Moresby and to the Bishops Museum in Honolulu, and afterwards, I will also continue to the American Museum in New York. Thus, all of the word’s museums that host the most important and most relevant collections of non-volant New Guinean mammals will be covered and the morphological part will be finished. So, once the genetics is done, and the morphology is done, I will hopefully know exactly and precisely what species I'm working with, what lives up there in the mountains of New Guinea. And then I can come back at the very beginning to the ecology, to the important questions, such as community ecology of the species along the mountain ranges, along the elevation gradients, and diversity distribution, species turnover, and similar really highly relevant questions, and also to describe some new species that emerged on the way. That's all from me and I would like to thank to all of my supporters for these wonderful opportunities.