Gubernatrix cristata, the Yellow Cardinal, is a rare bird species from the Pampas grassland. It has a restricted geographical distribution in southern South America and is alone to this Biome ( Uruguay, Argentina, and Southern Brazil ) . Fourteen field trips were made from April 2006 to March 2009 in an effort to happen persons and collect samples for familial analysis. A sum of 72 xanthous cardinal samples was taken in this survey, 59 were from modern-day specimens and 13 samples were from museum specimens. We accessed the species familial diverseness through 10 polymorphous atomic microsatellite venue of Gubernatrix cristata and a ND2 mtDNA fragment. We found merely three haplotypes with nucleotide and haplotype diverseness peers to Iˆ = 0.00277 and Hd = 0.6219. The Fst ( 0,00340 ) and Nm ( 73,18 ) shown a weak structuring with small familial distinction. Fu ‘s Fs statistic was significantly different from nothing ( 2,248 ) and Tajima ‘s D was positive ( 2,17506 ) . Both trials showed likely grounds bespeaking a lessening in population size and/or equilibrating choice. Analyses utilizing the “ no alloy theoretical account ” and a larger burn-in ( 500000 ) yielded no bunch of persons. Due to this consequence no single assignment to any geographical country was possible. The present survey aids in understanding the preservation needs for Yellow Cardinal by supplying information on the familial diverseness and population structuring along its distribution scope. Although we found no structuring within our survey country, farther survey is needed, analyzing the familial diverseness and population structuring throughout the species ‘ scope, adding more samples from wild animate beings from La Pampa population and Corrientes.
Keywords: Endangered species, Genetic diverseness, Gubernatrix cristata, Mithochondrial DNA, Microsatellites, ND2 cistron, Pampas grassland.
Gubernatrix cristata, the Yellow Cardinal, is a rare bird species from the Pampas grassland. It has a restricted geographical distribution in southern South America and is alone to this Biome ( Uruguay, Argentina, and Southern Brazil ) ( Azpiroz, 2003 ) . This species has undergone a dramatic population diminution across its scope and its preservation position is defined as endangered ( BirdLife International, 2000 ; Fontana et Al. 2003 ) . In Argentina, it is now rare except really locally ( Fraga, 1997 ) . In Uruguay, it was historically known from 13 sections, but late from merely Paysandu , Rio Negro, Florida and Rocha ( BirdLife International, 2000 ; Azpiroz, 2003 ) . In Southern Brazil ( Rio Grande do Sul State ) it was already considered rare in the 1970s and 1980s ( Belton, 1985 ) . Since so it suffered a dramatically decline and now is believed it is confined to restricted musca volitanss in the SE hills, near Uruguayan boundary line and at W tip of Rio Grande do Sul ( Fontana et al. 2003 ) . Once the deficiency of xanthous central sightings in the last few old ages and its bad state of affairs in the adjacents countries in Uruguay, the SE hills population seems to be isolated or even nonextant. ( Martins-Ferreira, 2007 ) . The staying populations are little and stray ( BirdLife International, 2000 ) . The lone genteelness wild population known in Brazil of G. cristata seems to be limited to Espinilho State Park ( Parque Estadual do Espinilho ) and milieus ( Damiani et al. , 2009 ) . The devastation and atomization of its home ground have been enumerated as possible extra menaces for the species, every bit good as the utmost force per unit area of gaining control to provide the traffic of wild animate beings ( Chebez 1994 ; BirdLife International, 2000 ) . The chief ground behind the traffick is that bird is a colourful and cantabile passeriform bird. The male has a bright xanthous thorax and back with black crest, oculus line and pharynx. It has the full supercilium and malar band yellow while the female are white with a grey chest ( Belton, 1994 ; Sick, 1997 ) . During the genteelness clip the male defends its district sharply atacking other males. That behavior bends to be a manner how poachers capture them utilizing a confined male as steerer ( Fontana et al. 2003 ) . Hudson ( 1920 ) already atested that its qualities as beautiful bird with loud and musical voice made it a favorite coop bird. The turning record of loanblends among xanthous cardinal and common diuca finch ( Diuca diuca ) in Argentina seems to be a response to the deficit of male persons in the population of the species ( Bertonatti & A ; Guerra, 1997, 2001 ) . With the void hypothesis that Yellow Cardinal has non a population construction throughout its distribution scope, we decide to look into that. Our purpose was to find the population position of Gubernatrix cristata across its distribution scope, taking in consideration the population construction, familial diverseness and cistron flow. Merely with such information in manus we would be able to suggest a specific preservation scheme to the species.
Fourteen field trips were made from April 2006 to March 2009 in an effort to happen persons and collect samples for familial analysis. In Rio Grande do Sul ( the southernmost Brazilian province ) , merely four Yellow Cardinals were found in a individual vicinity ( Espinilho State Park ) . In Uruguay, four separate field trips to different vicinities found merely six persons in Minas, Lavalleja Department, five in one farm and one in another land. All of them were pealing banded with rings provided by CEMAVE/ICMBio ( National Research Center for the Conservation of Wild Birds ) . In Argentina, a visit was made to the Museo de Ciencias Naturales Bernardino Rivadavia from which tissue samples of eight teguments were collected, one from each state where the species has ocurred, and one from Uruguay. Five more samples came from museum specimens: one from Museu de Zoologia district attorney Universidade de Sao Paulo ( MZUSP, Sao Paulo, Brazil ) , two from Museu de Ciencias Naturais district attorney Fundacao Zoobotanica do Rio Grande do Sul ( MCN/FZBRS, Porto Alegre, Brazil ) and two from Royal Ontario Museum ( ROM, Toronto, Canada ) ( Fig. 1 ) . The remainder of the samples came from specimens seized by the Environmental Police and are of unknown beginning. A sum of 97 xanthous cardinal samples was taken in this survey ( Appendix A.1 ) . Some of which holding been maintained ( but non bred ) in imprisonment by menagerie, preservation establishments, or private persons. Of these, 25 presented a hapless DNA quality that was n’t possible to continue with any analysis. From 72 samples that worked decently 59 were from modern-day specimens. The other 13 samples were from museum specimens ( old ages 1905-1975 ) selected from across the scope, but with a particular accent on the population in Argentina since many samples with unknown geographical informations were supposed to come. Blood samples were stored in FTA Cards ( Whatman ) and plumes were placed in 95 % ethanol solution. Museum tissues samples had their Deoxyribonucleic acid extracted utilizing the DNeasy Tissue Kit ( QIAGEN ) with the appropriate protocol harmonizing to the maker for each type of tissue ( Mundy, N. I. et al. , 1997 ) . Contamination with modern DNA or PCR merchandises was monitored by including two extraction spaces in every extraction unit of ammunition and prevented by executing all museum samples extractions in a dedicated ‘clean ‘ research lab, kept free of good quality DNA and PCR merchandises.
Figure 1. Map demoing the points of gathered samples from wild persons or from museum teguments across G. cristata distribution scope. Polygon used with permission from Birdlife.
A microsatellite-enriched genomic DNA library of Gubernatrix cristata was constructed and 10 polymorphous atomic microsatellite venue ( with 2-bp repetitions ) were selected on the footing of polymorphism degrees and size of elaboration merchandises. All samples were genotyped utilizing a polymerase concatenation reaction aiming different venue ( Table 1 ) . Primers and PCR conditions are given in Molecular Ecology Resources Primer Development Consortium ( 2010 ) . PCR merchandises were purified, rhythm sequenced on both strands, and run on an ABI 3730 sequenator.
We extracted DNA from plume samples utilizing the Alkali protocol for DNA extraction and attempted to sequence several mitochondrial DNA ( mtDNA ) venue utilizing debauched avian primers from Sorenson ( 2003 ) to find if sufficient familial variableness was present for analysis. The control part, ATPase8, and ND2 were chosen, as these parts have proven utile in population familial surveies of birds ( Burg and Croxall, 2001 ; Sorenson, 2003 ; Oyler-McCance et al. , 2005 ) . We successfully amplified parts of the ND2, but were unable to magnify control part and ATPase8. Given the lower quality of DNA infusions from many samples we decided to magnify the cistron in two pieces and run four sequencing reactions ( both strands of each PCR merchandise ) . The primers used were ND2H 5 ‘ CCT TGA AGC ACT TCT GGG AAT CAG A 3 ‘ ( Tavares et al. , 2006 ) – ND2F_Passerine 5 ‘ CCA YCC ACG AGC YAT TGA AGC 3 ‘ ( new primer designed ) , and MetLTF 5 ‘ AAG CTA TCG GGC CCA TAC CCG 3 ‘ ( Tavares et al. , 2006 ) – ND2R_Passerine 5 ‘ GCC ATG CRT TGG TYA TGC TNG AG 3 ‘ ( new primer designed ) . Those primers were used to magnify a 349 base-pair part that contained the variable sites in ND2. We amplified DNA via the polymerase concatenation reaction ( PCR ) in 12,5 I?l reaction volumes incorporating 2.0 I?l DNA, 7.92 I?l H2O, 0.5 I?l ( 10 pmol ) of each primer, and 1.58 I?l PCR maestro mix, dwelling of a 1.25 I?l buffer ( 10X:100 mM Tris-HCl, pH 8.3, 500 millimeter KCl, 25 millimeter MgCl2, 0.1 % gelatin ) , 0.28 I?l dNTPs ( 10 millimeter ) , and 0.05 U Taq polymerase ( Amersham Pharmacia Biotech ) . Reactions were performed under the undermentioned conditions: initial denaturation at 94A°C for three proceedingss, followed by 36 rhythms of 94A°C for 45 sec, 50A°C for 45 sec, and 72A°C for 1 minute and 30 sec, and a concluding extension of 72A°C for 7 proceedingss. The PCR merchandises were so electrophoresed on a 1 % agarose gel incorporating ethidium bromide and visualized under UV visible radiation. Amplified sections were purified by striking sets from agarose gels and centrifugating each through a filter tip. The sequencing reactions were so electrophoresed on an ABI Prism 3100 automated sequenator ( Applied Biosystems ) . We sequenced in both waies and sequences were aligned and assembled utilizing ChromasPro V. 1.49 ( Technelysium Pty Ltd ) and aligned utilizing the CLUSTAL W algorithm with default options, implemented in MEGA 4.0.2 ( Tamura et al. , 2007 ) . Alliances were checked and edited by manus when necessary. The haplotype sequences were deposited in GenBank under accession Numberss HQ15712 to HQ15714.
Table 1. Gubernatrix cristata microsatellite venue used in this survey.
Size ( bp )
( CA ) 7
( AC ) 8
( CA ) 8
( CA ) 12
( AC ) 9 ( CA ) 7
( CA ) 7
( CA ) 9
( CA ) 7 ( AC ) 5
( CA ) 7
( CA ) 7
Significant goings from HWE: * P & lt ; 0.001
We assessed the constriction history of these populations utilizing Bottleneck v.1.2.02 ( Cornuet & A ; Luikart, 1996 ) . This plan was used to observe a heterozygote surplus for single populations, sing the two-phased theoretical account ( TPM ) of microsatellite mutant, a 70 % stepwise-mutation theoretical account ( SMM ) and 30 % infinite allelomorphs model ( IAM ) , and 1000 reproductions. Several other combinations of the SMM: IAM ratio were tested to set up the sensitiveness of these informations to the mutational mechanism. The Wilcoxon signed-rank trial was used to find if the allele frequence distribution for a population exhibited important heterozygote extra comparative to pattern outlooks. Bayesian constellating with package Structure v.2.3.2 ( Pritchard et al. , 2000 ) was used to delegate persons to populations ( K ) based on posterior chances where K is unknown. The figure of groups was set to 1-7 with 3 tallies per K. Posterior chances were calculated for all K conjectural populations. All analyses were based on 1,000,000 Markov Chain Monte Carlo loops following a burn in of 50,000 loops.
To mensurate mtDNA diverseness, both haplotype diverseness, Hd, and nucleotide diverseness, Iˆ , and their standard divergences were estimated utilizing DnaSP v5.10.00 ( Librado & A ; Rozas, 2009 ) . To prove for grounds of recent population enlargement we calculated Fu ‘s Fs ( Fu, 1997 ) and Fu and Li ‘s ( 1993 ) D* and F* statistics to compare with Fu ‘s Fs. Therefore, if Fs is important and F* and D* are non, it is an indicant of population enlargement, while the opposite indicates choice ( Fu, 1997 ) . We besides calculate Tajima ‘s D ( Tajima, 1989 ) . We used ARLEQUIN 3.11 ( Schneider et al. , 2000 ) and DnaSP v5.10.00 ( Librado & A ; Rozas, 2009 ) to execute these computations. We created a mismatch distribution of pairwise differences utilizing DnaSP v5.10.00 ( Librado & A ; Rozas, 2009 ) to compare the expected distribution for a population. The distribution tends to be multimodal when populations are at equilibrium and unimodal in instances of recent demographic enlargement or decrease ( Rogers and Harpending, 1992 ) . To diagrammatically expose the ascertained mismatch distribution compared to the expected distributions for populations in equilibrium and enlargement, we used Roger ‘s method of minutes ( Rogers, 1995 ) as calculated in DNASP. The topological relationship between the haplotypes was estimated utilizing the plan Network 22.214.171.124 ( hypertext transfer protocol: //www.fluxux-engineering.com ) with average fall ining attack, for the three informations sets ( Bandelt et al. , 1999 ) .
Allelic fluctuation for the 10 atomic microsatellite venue ranged between 4 and 14 allelomorphs with an norm of 7.5 allelomorphs per venue. The ascertained heterozygosity ( HO ) varied from 0.126 to 0.893 and the expected ( HE ) heterozygosity from 0.130 to 0.930, with agencies and standard mistakes of 0.6824 + 0.1144 and 0.7132 + 0.0272 severally ( Table 1 ) . Four venue exhibited a going from Hardy-Weinberg outlook that may be the consequence of a sampling prejudice. Since the species is rare and most of the stuff used was from ictuss made by the Environmental Police ( Table 2 ) , we can non find if all of the samples represent the same population.
Table 2. Geographic beginning and beginning of the samples processed in our survey.
Consequences of constrictions sensing utilizing Wilcoxon signed-rank and mark trials under Infinite Allele Model ( IAM ) , Two-Phase mutant ( TPM ) and Step-wise mutant ( SMM ) are presented in Table 3. In bottlenecked populations, the ascertained cistron diverseness exceeds the expected equilibrium cistron diverseness under the premise of mutation-drift equilibrium. The void hypothesis tested for heterozygosity surplus utilizing Wilcoxon sign-rank trial provided ( P & lt ; 0. 05273 ) , ( P & gt ; 0.09668 ) and ( P & lt ; 0.42285 ) chances under the IAM, TPM, and SMM severally. The void hypothesis is accepted under the SMM theoretical account merely, connoting that the Gubernatrix cristata has non experienced any recent familial constrictions. The estimated values of the heterozygosity surplus and their chances in gestural trial were 5.89 ( P & lt ; 0.15033 ) for the IAM, 5.90 ( P & lt ; 0.15148 ) for the TPM and 5.91 ( P & lt ; 0.38936 ) for SMM. The void hypothesis of mutation-drift equilibrium was non besides accepted based on the mark trial under mutant theoretical accounts.
Not all of the 97 sampled persons were successfully sequenced for the mark ATPase 8 or Control Region fragment. Merely 67 were succesfully sequenced for the mark ND2 fragment ( haplotype sequences were deposited at GenBank under accession HQ15712 to HQ15714 ) . A sum of 349 bp were aligned in G. cristata samples contained two variable and parsimony enlightening sites and a sum of three haplotypes ( H1 – H3 ) . Among H2 and H1 haplotypes 75 mutational stairss were found whilst 34 among H2 and H3 ( Fig. 2 ) . Estimates of base and haplotype diverseness in the sample were comparatively high, with Iˆ = 0.00277 and Hd = 0.6219. The genealogical relationships between haplotypes were estimated in the median-joining webs illustrated in Figure 2. The web revealed no construction form at all. Haplotype 2 had the highest chance of represent antediluvian signifiers because of its cardinal place. The Fst ( 0,00340 ) and Nm ( 73,18 ) shown a weak structuring with small familial distinction. The Fst analysis was done following Nei ‘s ( 1978 ) categorization where a value is considered low when Fst & lt ; 0,05 ; medium when 0,05 & lt ; Fst & lt ; 0,15, and high when Fst & gt ; 0,15. Lack of support to a demographic enlargement hypothesis was either obtained by different trials made to prove the impersonal theory of molecular development: Fu and Li ‘s D* trial statistic ( 0,71916 ) showed no statistical significance ( P & gt ; 0.10 ) every bit good non important was Fu and Li ‘s F* trial statistic ( 1,34814 ) ( P & gt ; 0.10 ) . Fu ‘s Fs statistic was 2,248 which was significantly different from nothing and is a likely grounds bespeaking a lessening in population size and/or equilibrating choice. Tajima ‘s D was positive ( 2,17506 ) what signifies low degrees of both low and high frequence polymorphisms, bespeaking a lessening in population size and/or equilibrating choice.
Table 3. Familial constriction sensing utilizing Wilcoxon signed-rank mark trials under Infinite Allele ( IAM ) , Two-Phase ( TPM ) , and Step-wise ( SMM ) mutants theoretical accounts of microsatellite development
Wilcoxon signed-rank trial:
Probability of Heterozygosity surplus
P & lt ; 0.05273
P & gt ; 0.09668
P & lt ; 0.42285
Number of venue with heterozygosity surplus
Expected ( chance )
P & lt ; 0.15033
P & lt ; 0.15148
P & lt ; 0.38936
Figure 2. Genealogic relationships between haplotypes estimated in the median-joining webs. H_1, H_2 and H_3 – Haplotyes ; 75 – mutational stairss between H2 and H1 ; 34 – mutational stairss between H2 and H3. Yellow – unknown geographical beginning ; Red – persons from Argentina ; Green – persons from Brazil ; Blue – persons from Uruguay
As the exact geographical beginning of many ( 45 % ) of the sampled Yellow central persons is unknown or unsure ( many of the persons sampled in menagerie or at genitalias, or got from constabulary ictuss were non associated to dependable information on their exact location of gaining control ) , the construction analysis would be peculiarly relevant because it would let bunch of the persons without the demand for a priori geographical information. Three independent tallies for one to seven populations utilizing an alloy theoretical account and correlated allele frequences among populations ( Falush et al. , 2003 ) , were run. Figure 3 shows the consequences for thousand peers to 3 populations as saloon secret plan and trigon secret plan. Analyses utilizing the “ no alloy theoretical account ” and a larger burn-in ( 500000 ) yielded really similar consequences ( informations non shown ) . The consequences for all tallies were practically the same proposing no bunch of persons ( Fig. 3 ) . Due to this consequence no single assignment to any geographical country was possible.
The mismatch distribution of ascertained haplotype fluctuation was unimodal uncovering a going from expected fluctuation under a Changeless population size ( Fig. 4 ) ( k = 0,965, discrepancy of K = 0,7205, Raggedness statistic R = 0,1319 ) .
Bar secret plan demoing no construction for K = 3 populations.
Triangle secret plan demoing no construction for K = 3 populations.
Figure 3. Consequences of Structure tally. Here is depicted when K = 3 populations. a ) saloon secret plans and B ) trigon secret plan.
Figure 4. Mismatch distribution of the ascertained haplotype fluctuation from 67 Yellow Cardinal mtDNA samples compared to the expected under a population enlargement.
Endangered birds normally have little and disconnected populations. A normally concern about population surveies is a proper sample size to rightly deduce familial diverseness steps. Despite the comparatively little sample size of this survey it has been recommended that at least 20 to 30 persons be sampled in microsatellite surveies ( Pruett and Winker, 2008 ) . Our 72 sample size is more than plenty to run surveies about familial diverseness and population construction appraisals. The findings about no structuring look to be related to other issues.
The deficiency of population construction showed by microsatellites with low Fst and big Nm seemingly tell us the history about merely one immense population across Yellow cardinal scope with an intense cistron flow. The same is truth for ND2 mitochondrial DNA cistron.
The fact that both Fu ‘s Fs and Tajima ‘s D indicates a lessening in population size and/or equilibrating choice shows us a possible account of what is go oning.
The xanthous cardinal has suffered an intense hunting force per unit area by about one hundred old ages now ( Hudson, 1920 ) . This clip period is non sufficient to be detected at the molecular degree, nevertheless is adequate to drastically cut down the species population size or even driven to extinct some subpopulations ( Martins-Ferreira, 2007 ) .
The species had a broad distribution country in the yesteryear that has been reduced over clip, both due to alterations in home ground, as the hunting force per unit area. Today it seems to be limited to a narrow uninterrupted irregular arc-shaped set in Argentina, from San Luis, go throughing through La Pampa, Cordoba, Santa Fe , Entre Rios and Corrientes ( Marchiori, 2004 ; Martins-Ferreira, 2007 ) . Following the halfway South of Uruguay to the west terminal of Rio Grande do Sul, Brazil. That decrease in its distribution scope to merely the optimum environment for the species, besides ended up cut downing their familial variableness and, perchance, the haplotype diverseness. The Whooping Crane ( Grus americana ) experienced a terrible diminution in their population size in early 1900. Reducing its population to 14 persons in 1938, resulted in the loss of two tierces of its haplotype diverseness. Merely three haplotypes were identified in the current population ( Glenn et al. , 1999 ) .
As shown in Whooping Cranes, a terrible decrease in population size can do a familial constriction, ensuing in loss of familial diverseness ( Nei et al. , 1975 ) . However, the impact of a constriction on population familial diverseness depends on how quickly the population declines, the size of the constriction population and the continuance of the constriction. When a population suffers a familial constriction, rare allelomorphs ab initio are lost, but the population can keep diverseness if the population recovery is rapid ( Allendorf, 1986 ) . If population copiousness remains low, diverseness can go on to be lost due to familial impetus ( Allendorf,1986 ) . There are several possible accounts for the comparatively high familial diverseness observed in Yellow Cardinals along Southern South America. First, the Yellow Cardinal diminution may hold been less terrible than that of the Whooping Crane. The population position in the early 1900s is unknown, but ne’er drop low earlier than today. Second, population recovery may hold been sufficiently rapid so as non to hold lost diverseness due to familial impetus. Although Yellow Cardinal in Uruguay and Brazil has late experienced several episodes of population diminution, each clip population recovery appears to hold been rapid. Finally, recolonization by persons that emigrated from countries where familial diverseness remained high, such as Argentina, could hold contributed to the high familial diverseness. The ascertained familial similarity within the part is consistent with an unfastened population, as cistron flow is expected to homogenise populations ( Charlesworth, 2003 ) . We detected no form in the distribution of haplotypes to propose that familial structuring exists in Yellow Cardinals along its scope.
Therefore, our consequences back up pull offing Yellow Cardinals along its distribution scope as a individual genteelness population. It is possible that the Yellow Cardinal may organize one big panmictic population. The being of one big population could explicate the high degree of familial diverseness found in the population by leting the effectual population size to stay high during periods of population fluctuation and service as a beginning of immigrants. Although the Yellow Cardinal ‘s demographic history does non demo drastic decreases in familial diverseness, the population size fluctuations appear to hold had no noticeable effects on the distinction and frequence of haplotypes as evidenced by the mismatch distribution and Fu ‘s Fs. The minimal spanning web ( Fig. 2 ) shows that the 3 haplotypes are non closely related ( within 75 and 34 mutational stairss ) . A triangle-shaped evolution, as shown in Fig. 2, state us merely that a population has experienced a terrible diminution on its familial diverseness. The Yellow Cardinal population still appears smaller than historical histories suggest. Historical histories described Yellow Cardinal as common in Argentina in the 1900s ( Hudson, 1920 ; Wetmore, 1926 ) . Therefore, it seems that the population may hold being enduring a diminution in its Numberss from the last century until now.
The present survey aids in understanding the preservation needs for Yellow Cardinal by supplying information on the familial diverseness and population structuring along its distribution scope.
Although we found no structuring within our survey country, farther survey is needed, analyzing the familial diverseness and population structuring throughout the species ‘ scope, adding more samples from wild animate beings from La Pampa population and Corrientes.
The chief deduction for Yellow Cardinal preservation with these consequences is that a confined genteelness plan could be established on an international degree, without much concern about continuing specific haplotypes, since all populations portion the same 1s.
We thank to Oliver Haddrath for the enormous aid in carry oning the lab work at ROM. We thank to Carla Lopes, Paula Rorato, and Nelson Fagundes for assisting us with the package bundles and valuable treatments. We thank to Darlise Lopes for the last-minute aid in the lab. We thank CNPq for supplying the doctor’s degree scholarship to CMF, to CAPES for the Sandwich Scholarship, and to FBPCN for financing the undertaking. Laboratory work was funded by NSERC runing grant to AJB.