Species identification of Australian Museum black corals

Hi everyone, my name is Jeremy Horowitz and I was awarded the Australian Museum Foundation AMRI Visiting Collections Fellowship this year and it allowed me to take a look at the black coral collection at the Australian Museum and the title of this talk is 'Shining a light on dark taxa: an integrated approach to species identification of Australian Museum black coral collection.'

And I should preface this talk by saying we know so little about black corals in the world especially in Australia because most of the world's taxonomic experts live in the States. So this was an amazing opportunity to to learn more about black corals in you know this part of the world. My affiliations are James Cook University the Centre of Excellence for Coral Reef Studies, the Museum of Tropical Queensland and the Australian Museum Research Institute.

A little background about why we know so little about biodiversity. So anthropogenic drivers are threatening biodiversity on a global scale. We don't know how many species live on earth somewhere between you know half a million and 10 million species live on earth. And what's worse is that we don't know what we don't know and by that I mean you know studies have estimated somewhere between three percent and 96 percent of species aren't yet even described. And what that means is that we know very little about biodiversity and it also makes it really challenging to create conservation interventions to you know target areas or specific species that need the most protection.

So why do we know so little about biodiversity? Obviously this talk will be in the marine realm. We've been scuba diving, it's been mainstream for about 75 years so between, you know between zero and about 20 meters depth most people can dive down and so we know a lot about the shallowest of marine habitats. In the last few 50 years or so, we've created new technologies to dive hundreds of meter. So this is a drawing of a closed circuit rebreather and so it allows you to recirculate your air so you could stay longer and dive deeper. And we also now have the ability to go on research vessels and use remotely operated vehicles which can dive thousands of meters. But of course there aren't many ROVs in the world, especially ones that can dive down really deep. And you need a high level expertise to scuba dive yourself down just 100 meters so most of what we know is this first 20 meters of depth.

And so a question I had really early on when I started studying black corals is what proportion of the marine habitat is represented by this 20 meters of depth? So I took the depth at every latitude longitude degree. I'm using a bathymetric map and I made a histogram. And just with depth on the x and percentage of ocean coverage on the right with bins at 20 meters. That first bar represents zero to 20 meters of depth. And so what this tells us is we know a lot uh about one percent of the ocean and this is the rest. So we don't know much about biodiversity for the majority of the ocean.

All that we don't know about biodiversity are known as these biodiversity shortfall. And there's more than three but the three main ones are the ones that I've worked on are the Linnaean shortfall, which represents our lack of knowledge about known species. So this is identified, named after Carl Linneaus who formalised binomial nomenclature. And so he described how we go about describing species. The Wallacean shortfall which represents our lack of knowledge about species distributions.

And that was named after Alfred Wallace who independently came up with the theory of evolution through natural selection and he also prompted Darwin to publish on the Origin of the Species. And the Darwinian shortfall which represents our lack of knowledge about species and molecular relationships and evolutionary histories of species. And of course that was named after Charles Darwin who's the first person to think about evolution in terms of a tree and it's how we think about evolution today.

It's really important to identify, to understand biodiversity better because it enables us to identify these ecological and evolutionary processes that maintain biodiversity patterns. And these are really important to identify because then you could target those specific processes and design conservation interventions to protect those processes and therefore preserve biodiversity.

If we want to go out and learn more about what's living in the ocean it's really challenging to just go out and you know collect representatives from all over the world or even within an area like the Great Barrier Reef. But what you can do is go visit a museum collection. So they're an inexpensive alternative. They often represent biodiversity of the given region. They're often preserved in some way where you can extract DNA from it and each jar contains documentation of the history of the specimen, so you know who described it, where it came from, the species ID and you know how the species ID has been updated through time. And lastly this is also the location where most holotype specimens live. I'm probably preaching to the choir but a holotype specimen is the specific specimen from which a species description was based. And so it's really important to refer back to these holotype specimens when either describing new species or identifying a specimen to the species level.

The Australian Museum Research Institute has over 20 million specimens collected over the last 190 years. I did a quick Google search and one of the largest coral collections of Australian fauna in the world. And they even have over 200 black coral samples representing 130 year collection. And looking at a printout of the specimens in the museum collection before my visit, half of the specimens weren't identified to the family level and 30 specimens were identified to the species level. And I said it before but we know so little about black corals in Australia that every collection, especially one that is large and well preserved as this, represents you know a treasure trove of biodiversity information.

And so therefore we have the opportunity here to update knowledge about biodiversity ranges to and within Australian waters. Sorry about that. Black corals, okay. So black corals, in the order of Antipatharia are found...I think I lost my. You're still sharing your screen but maybe if you just, yeah there you go. I'm back, okay. So black corals are found all over the world, in all habitats from depths from just above the surface, which surprises some people to over 8000 meters deep. They can be really large and branching like in the shallow waters, like in the first image. They could be really small and intricate like in the second image. They have polyps or feeding mouths, in the third top and bottom images. They could be really small polyps or really large and they have these characteristic skeletal spines that are really informative in identifying species. But of course they're really small so there's less than a millimetre tall and so you need a scanning electron microscope to view and measure these really informative features. To place black corals in the Anthozoa, black corals are Hexacorals. People often confuse black corals with soft corals which are in Octocorallia but black corals have six tentacles and soft corals have eight tentacles. Previously, it was thought that black corals were really closely related to the tube anemones.

So much so they even created an entire group just for these two orders called the Cerantipatharia. But we now know that black corals are related to hard corals and the Corallimorphs and the actinarians are sister to the black corals Corallimorphs and Scleractinians as one group. It's really hard identifying a black coral species and that's because they don't have, they have a few morphological features but there's lots of overlapping features between species, genera and even families. And what was previously thought as an informative feature was whether coral branches or if it's unbranched or whip. So we have a picture outlined in blue, that's the branching black coral and then the red one's the whip black coral. And behind is a phylogenetic tree that depicts molecular relationships between species.

And this is what was assumed before we started sequencing species, where all the whip corals would be in one clade or in one natural group and branching black corals would be separate. But after sequencing a whole bunch of species, this is more of what we found. And so you can see these whip corals popping up in different parts of the tree. And what this tells us is that, well one it's not an informative feature in separating species genera or even families. There's two different families here. But yet we don't know. What it means is that the whip coral morphology has evolved multiple times over evolutionary history and it's not an informative feature.

So what I've been doing for a big part of my PHD is getting actual specimens either collecting them or using museum specimens. Identifying them morphologically and sequencing them to get some molecular data. And then use those two lines of evidence to identify specimens of the species level. And what this has enabled me to do is increase our knowledge about the numbers of species that live in Australian waters, expand the ranges of species and also describe a ton of new species.

And I'll do a little more specific information about the methods here. So morphological identification; I use three different features generally, although at the species level, certain features are informative. One is the gross morphological features, so whether branches or whether it's unbranched, it's not too informative. But also whether it has branches or what we call pinnules which are repeated branches along the stem or prior branch. And if you think of a fern and a fern has pinnules.

And the polyp characteristic; so the tentacles surround the feeding mouth and the size of the polyp, the distance between two polyps, the orientation of the tentacles around the polyp, the length of the tentacles. So these are all different little morphometrics that we could use to separate species.

And as I mentioned, as I mentioned before, the spines are a really informative feature. So the middle image is a zoomed out view of a skeleton using a scanning electron microscope. And on the corners are different species and so you can see even though a coral may look really similar from a gross morphological perspective, when you look at the spines they can be really different and really useful in doing taxonomy.

The molecular method that I've been using for my PHD are ultraconserved elements. I'll give a brief background on UCEs. So as the name implies, they're ultra conserved and what that means is that these regions of the genome evolve so slowly through hundreds of millions of years that you could actually match up these identical regions among highly diversion taxa.

Obviously comparing identical regions of the genome isn't going to be very informative; they're going to say they're all the same. But on the image on the right, you could see as you move away from these UCE regions towards what we call flanking regions, variability in base pairs increases.

So we grab the UCE and flanking regions, line those up and we can detect differences between species. And what's useful about UCEs compared to a single locus marker is that there's thousands of UCEs in a given genome. So that means we have thousands of loci, thousands of alignments to use, to provide really strong bootstrap support in our resulting phylogenies.

So we had two weeks at the Australian Museum Research Institute and the main aims of this project were to one, digitise the collection. We spent most of the two weeks really just going through the collection and you know just sorting them and seeing what was in there. So we now have a digital collection, so if someone at a later date wants to look at the black corals all they need to do is look at wherever the digital collection is stored. Identify the specimen as morphologically, subsample skeletons for scanning electron microscopy and also self-sampling tissue for UCE sequencing. And using that information to identify the species and update what we know about biodiversity in Australia.

Step one was to open the jars. So as I mentioned, this is a really old collection and some of the samples have been unopened since they were first jarred. So this sample is from 1911 and it most likely hasn't been opened since then. So Steve and I, we had to crack a few of the jars just to open them.

And so we went through the collection and took out samples one at a time and we imaged each sample. We then described the morphological features as I mentioned, the gross morphological features, the polyps and the spines were looked at later.

And so we had a table of every specimen, its morphological features and its associated image. And next step was the subsample for sequencing and also for SEM, for taking SEM, so we have an SEM here at Museum of Tropical Queensland. So it took weeks to take SEMs of all the specimens that we wanted to SEM. So it made more sense to bring the samples back here and then spent, it's been a month now of scanning the 90 to 100 samples that we've subsampled.

And so here's the SEM in MTQ and so Savannah on the left and Abba on the right were really helpful in assisting with taking all these SEMs. And so here's some of the overall results to date. So out of the 210 specimens that we looked at, 126 had family and genus level updates. It's a pretty big overhaul of you know what we thought we knew about the collection. And then two weeks later and 90 specimens were subsampled for SEM and UCE and we've now updated, we've now taken SCMs of all these 90 samples, and we're working on identifying the species. I'm finishing my PHD in December so I'm trying to park my time on these two different projects. But early next year I should have more species names. And also we're sending off DNA for sequencing. And so that means early next year, we're going to have morphological IDs based on the morphologies and also really good sequence data to match up to already sequenced specimens to have a lot of information to pin a species name or describe a new species in this collection.

Here are the some of the really cool highlights that we found over these two weeks. So as I mentioned, soft corals and black corals are often confused for one another and in this collection we found a bunch of soft corals were actually black corals and black corals are actually soft corals. So here's a sample that was initially identified as a soft coral and we, well we looked at it and identified as a black coral so we moved it over to the black coral section of the wet lab. We took sub-samples of this specimen and we took SEMs. Here's an SEM of the sample. And soft and black coral things identified as black corals that were actually soft corals. So what we were able to do is take it out of the black coral section of the of the store and put it in the soft coral area for a soft coral expert to work on.

When first going to the Australian Museum, I really wasn't expecting to actually work on any holotype specimens but we actually found two in the collection which is really exciting. This is a species in the genus Cirrhipathes and here's the actual full colony. It's really well preserved, really big and so I'm standing on the ladder here and you can see the inset image. You can see that it has its polyps and tissue like perfectly preserved. So we took a subsample and we'll try to sequence this old holotype from from 1889.

So here's the original description. Here's a figure in the description that shows the skeletal spine features and it's a great drawing, but it's really hard to tell what the spines look at or ornamentation that's on the spine. So we were able to for the first time, take a look at the spines of this holotype specimen and this is what it looks like. So you can see that it has ornamentation on from the tip to about midway down each spine and that's a really important feature that probably wasn't very clear in the original description. And so now we could do is well one, redescribe the species with this new information and sequence data. But two, look at other whip corals that have also been described that might actually have these exact same features and we could then you know synonymise them if they're the same species. We also found new species, which is always very exciting.

I've done, I've worked in a few different museums providing curatorial assistance and every museum I come across there's always undescribed species and the Australian Museum is no different. So this specimen was collected by Penny from the Tasman Sea at 500 meters depth. And you could see if you look closely, you could see that the branches or we call pinnules, they're alternating. And to us, that will tell you that it probably belongs in the Alternatipathes and the genus Alternatipathes. And we took a skeletal spine image which is that second image and so the spines would suggest that it's a new species but really the gross morphological features really scream it's a new species.

So here's an image on the left of the typical Alternatipathes that among the described species in the genus and all of the species have these really long unpinnulated branches, stems, sorry, and in this new species the stem is really quite short. So that was the first indication that was a new species. But then looking at the spines, the spines are much more rounded than the other species in this genus so we have a lot of evidence to support it describing it as a new species. But we'll also try to sequence the specimen.

There's also these really cool abyssal adaptations that I just wanted to talk about. I think some of you are interested in. So most species in the order have these basal plates and they attach to hard substrate. But in the collection we actually found a species and it's one of three species that don't have this basal plate. Instead they have this basal hook and this basal hook allows it to actually settle and grow where hard substrate isn't present. That's a really cool adaptation because this is an abyssal species, so it makes sense where there's very limited hard habitat for these kinds of adaptations to occur.

But what's interesting is that I've recently time calibrated one of my phylogenies including this lineage and it's only 30 million years old. And you may think that's a long time, but black corals have been around for over 400 million years. So we also know that black corals originated in the shallows, you know shallower than a thousand meters depth. And this is a specimen that's commonly found in the abyss, you know in up to four or six thousand meters. So what this tells us is that it's taken about 400 million years for black corals to invade offshore heading towards the abyss. And it's taken that time probably because they've had to modify or adapt with these abyssal morphological features in order to survive where nutrients are less and notably here where there's very limited hard substrate. And so this is probably a recent, this is probably indications of you know more species diversify in this habitat, now that it's established populations in the deep. And we even we even found this exact same species in Western Australia. So we were on a trip in Cocos of Christmas Island with some of the Australian Museum researchers and we found that really beautiful Schizopathes sp. which is what this is so we now know that it actually occurs not only in the GBR in coral sea but also on Western Australia as well.

So some of the summaries, this fellowship allowed me to update species identifications, which is really always very good for a group that doesn't have a lot of work on it. So we're able to update knowledge about species living in Australian waters and their ranges. Excitingly we will also be able to redescribe species that were described in the late 1800s, which is really important to do. And that we now have new technology to better understand what a species is and how it relates to other species. And of course we're going to be describing a whole bunch of new species from this collection specifically. And we'll also sequence the specimens to understand how they're related molecularly. So generally we're going to update knowledge about biodiversity and taxonomy of black corals in Australia and the whole purpose of this is to have a better understanding about biodiversity, you know not just in the shallows but also you know, on the slope and in the abyss and to give managers more information to make informed conservation decisions when the time comes to better preserve our biodiversity.

I want to thank Steve, Laetitia, Ingo, Penny, Elena, Meagan and Claire, they made me feel really comfortable. Christina and I, when we were at the Australian Museum and also my MTQ team, Savannah and Aahba for helping with SEMs and also helping work on some of the specimens. And Tom Bridge and Peter Cowman and the rest of the MTQ team and lastly of course the AMF/AMRI Visiting Collections Fellowship. Thank you for funding this research.