1. Barcoding Neotropical Psittacidae
ABBI Regional Working Group Meeting
Buenos Aires, March 2007
Cristina Y. Miyaki
Departamento de Genética e Biologia Evolutiva
Instituto de Biociências
Universidade de São Paulo
I’d like to thank Pablo Tubaro and all his team for organizing this meeting.

2. Psittaciformes: distribution
The Psittaciformes occur in the American continent, in Africa, in south Asia and in the Australian region. All the species and genera that occur in the Neotropical region are endemics. Around half of the species and one third of the genera occur in the Neotropical region. All these species are grouped in the tribe Arini.
150 species [total in the Order: 330] (del Hoyo et al. 1997)
30 genera [total in the Order: 84] (del Hoyo et al. 1997)
Notropical Psittacidae or tribe Arini (Smith 1975)

3. Nestor notabilis
Amazona farinosa
Graydidascalus brachyurus
Pionus maximiliani
Anodorhynchus hyacinthinus
Ara ararauna
Orthopsittaca manilata
Primolius auricollis
Nandauys nenday
Diopsittaca nobilis
Guarouba guarouba
Enicognathus leptorhynchus
Rhynchopsitta pachyrhyncha
Hapalopsittaca amazonina
Pionites melanocephala
Bolborhynchus lineola
Nannopsittaca panychlora
Myiopsitta monachus
Forpus crassirostris
Chalcopsitta cardinalis
Pseudeos fuscata
Trichoglossus haematodus
Lorius garrulus
Melopsittacus undulatus
Neophema elegans
Agapornis roseicollis
Eclectus roratus
Psittacula krameri
Poicephalus senegalus
Psittacus erithacus
Cacatua galerita 99
100 98
Monophyly of Arini
Bayesian unrooted (3062 bp 12S, 16S & intron 5 tropomyosin)
Red = Neotropical
Blue = African
Green = Australasian
Bold: no MP support, only Bayesian
This is data from our lab that shows that there is strong support to believe that the tribe Arini is a monophyletic group. Other reserachers found the same result.
Matsumoto et al, prep.

4. Arini 150 species 27% endangered
Photo: P.J. Faria
Arini
150 species
27% endangered
The Neotropical genera show great variation in morphology (color, size) and ecology. Around 27% of the species are considered endangered in various levels. For instance, the Spix’s Macaw was declared extinct in the wild very recently. The main threat is habitat destruction, but this group is also affected by capture in the wild for the pet trade.
Source: Forshaw 1989

5. Species identification of non hatched embryos
Application of DNA barcodes in Neotropical Psittacidae
Species identification of non hatched embryos
Photo: Folha on line
Recife airport: to Lisbon
58 eggs (possibly parrots)
Incubator: non ideal conditions
Visited many places in Brazil (Amazon, Mato Grosso, Tocantins and Brasília)
And we believe DNA barcodes can help in specific problems that affect the conservation of this group. One example is here, the identification of species of unhatched embryos. This is an international smuggler who was caught before he left Brazil. He was carrying 58 eggs around his waist so that it would keep the eggs heated and the embryos alive until his final destination. However, as he was caught, the eggs had to be artificially incubated and he deliberately modified the conditions so that all the embryos died. The external analysis of the eggs suggested that they were from parrots. As the smuggler had been in many places within Brazil, the eggs could belong to various species. So the question was: which species?
Which species?

6. Species identification of non hatched embryos
Choice of marker: mitochondrial 16S rDNA
Sequenced bp: 6 groups (haplotypes)
Analyses: BLAST (GenBank) and distance
Samples from the embryos arrived in our lab and we chose the marker for which we had the largest dataset of parrot sequence available at the time: the 16S ribosomal DNA. We sequenced all the embryos and obtained short sequences that could be grouped by their identity (we had six groups of identical sequences). We did a BLAST analysis against the GenBank and a distance-based analysis. The results were totally congruent, so I am only going to show the distance tree.
Matsumoto, Oliveira-Marques, et al., in prep.

7. 213 bp of 16S rDNA Ara ararauna (3) Salvatoria xanthops (50)
Amazona aestiva/ Amazona ochrocephala (4)
First of all, I’d like to emphasize that this is NOT a phylogeny. The three sequences from group 2 clustered with Ara ararauna. The 50 sequences from groups 3 to 5 clustered with the monospecific genus Salvatoria xanthops. The four sequences from group 1 clustered with the species complex Amazona aestiva/Amazona ochrocephala. Only one sequence was not a psitacid. In this analysis we had a broad representation of the genus Amazona, thus we continued our analysis focusing in the identification of group 2.
Non psitacid (1)
Matsumoto, Oliveira-Marques, et al., in prep.

8. 390 bp of 16S rDNA Ara ararauna (3) Ara macao Ara chloroptera
Ara militaris
Ara ambigua
Primolius maracana
Primolius auricollis
Primolius couloni
Guarouba guarouba
Diopsittaca nobilis
Orthopsittaca manilata
Ara ararauna
Group 2
Ara glaucogularis
Aratinga cactorum
Ara severa
Cyanopsitta spixii
Anodorhynchus hyacinthinus
Here we have all the species from the genus Ara. Once again, this is NOT a phylogeny. Once again, the sequences from group 2 cluster with Ara ararauna.

9. Species identification of non hatched embryos Conclusions
The available 16S rDNA data base was informative to identify the species of the embryos.
The species identification was confirmed in another lab that used another mitochondrial marker (cytochrome b).
Would COI be a good marker?
Building a COI database for Neotropical parrots and will test the same embryos.
Thus, from this example we can conclude that we were able to identify the species of the embryos using a 16S database. Also, another laboratory identified the same species based on cytochrome b.
Now, our question is: is the COI a good marker too? To try to answer this question, we are sequencing samples from Neotropical parrots and will test the same embryos.

10. COI sequence database for Neotropical parrots
Master’s Degree student: Priscila F.M. Gonçalves (Dec 2006)
Objective: construct the database and evaluate its usefulness for species identification (e.g. evaluate if genetic distances within and among taxa do not overlap)
This is the Master’s Degree project of a student who started to sequence last December. The objectives other study are to build a database of COI sequence for Neotropical parrots and evaluate its usefulness for species identification.

11. COI sequence database for Neotropical parrots
Samples
113 out of 150 species of Neotropical parrots (34 with skins)
826 bp
980 bp
LTyr
Bird R1
COI H7557
Primers
LTyr (O. Haddrath)
Bird R1 (Hebert et al 2004)
COI H7557 (P. Gonçalves)
We have samples from the majority of Neotropical parrots. As many of them are endangered, we do not have many linked to museum skins. Initially the primers LTyr and Bird R1 were used for amplification, but as the resulting sequences were not very good at the 3’end (Bird R1), we designed a third primer (COI H7557). Now, we’re using LTyr and COI H7557 for amplification and all the three primers for sequencing.

12. COI sequence database for Neotropical parrots
PCR conditions (touchdown)
5 min 95ºC;
14 cycles: 30 sec 95ºC, 30 sec 68ºC (1°C lower per cycle), 40 sec 72ºC;
29 cycles: 30 sec 95°, 30 sec 54°C, 40 sec 72°C;
10 min 72°C
Sequences obtained: 23 (March 2)
Average size of sequences: 792 bp ( bp)
The PCR conditions are standard. We only have 23 good quality sequences. As the protocol is now standardized, we hope to work faster. The average size of the sequences is 792 bp.

13. Acknowledgements Erika Tavares, Royal Ontario Museum, Toronto
We do not have specific funding for the DNA barcodes project, but funds come from other larger projects and we rely on several collaborators as IBAMA.
Erika Tavares, Royal Ontario Museum, Toronto